Journal of Comparative Physiology A

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

We investigated the hearing mechanism of Manduca sexta caterpillars to determine whether their behavioural responses to acoustic stimulation are caused by sound-induced substrate vibration or by direct detection of airborne sound. Behavioural response thresholds to direct substrate vibration and airborne sound were measured using pure-tone stimuli at 150 Hz and 2000 Hz. A frequency of 150 Hz was selected based on previous behavioural measurements showing the lowest sound detection thresholds between approximately 100 and 200 Hz, while 2000 Hz was selected based on laser Doppler vibrometry measurements that revealed a mechanical resonance near this frequency in a thoracic hair. At both test frequencies, the direct substrate vibration detection thresholds were more than 100 times greater, on average, than the sound-induced substrate vibrations measured at the behavioural sound detection thresholds. Thus, the behavioural responses to airborne sound cannot be explained by sound-induced substrate vibration. During airborne sound stimulation, caterpillars exhibited graded behavioural responses with increasing sound pressure level, progressing from freezing to twitching and finally to jump-startle. Together, these findings provide direct experimental evidence that the behavioural responses of M. sexta caterpillars to acoustic stimulation cannot be explained by sound-induced substrate vibration and instead support direct detection of airborne sound. Understanding how caterpillars respond to airborne sound may also provide biological inspiration for the development of bio-inspired acoustic sensors.

Increasing experimental evidence suggests that biological systems cancel predictable components of sensory signals while maintaining sensitivity to externally induced state changes. This strategy provides task-specific sensor responses for posture, locomotion, and gaze control. A prime example is found in interneurons that respond to visual image shifts resulting from the relative motion between an animal's eyes and its visual surroundings. Such optic flow-processing interneurons, found across phyla and are particularly well characterized in Dipteran and other flying insects. We studied optic flow-processing interneurons in the Monarch butterfly whose large and highly contrasted wings sweep through the visual field with every wing-beat cycle, potentially obscuring interneuron output signals. Our results show baseline spiking activity increases when animals flap their wings, and individual spikes are phase-locked to the wing-beat cycle, even in the dark, when no visual motion input is available. A qualitative estimate of the interneurons' response to directional wing motion through its receptive field is not sufficient to explain the recorded activity patterns. Our results suggest that an additional internal signal suppresses responses to wing-induced visual motion to support effective vision-based stabilization reflexes. These findings support the principle that self-generated signals are suppressed while sensitivity to external modulation is preserved.

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.

Over the course of evolution, many species have adapted to the environmental conditions found at higher latitudes. This is likely also true of the circadian system, which regulates daily behavior and helps measure day length to facilitate adaptation to the seasons, though how the circadian clock adapts to latitude remains largely unknown. Here, we investigated daily activity patterns under long photoperiods and in constant darkness, as well as the underlying circadian clock network, in 52 Drosophila species from around the world. We found a strong, significant positive correlation between the clock's plasticity, measured as the phase difference between morning and evening activity, and the average geographical latitude from which the species originate. The mechanisms underpinning this increase in plasticity differed between species: in the subgenus Siphlodora, the clock weakened with increasing latitude, accompanied by a reduction of the neuropeptide Pigment-Dispersing Factor in the small ventrolateral clock neurons (s-LNv), whereas in the subgenus Drosophila the opposite was true. The subgenus Sophophora, by contrast, showed a negative correlation between circadian plasticity and PDF levels in the s-LNv axons. These results suggest that Drosophila species have evolved diverse, independent strategies to adapt their circadian clock to higher latitudes.

Foraging decisions often depend on net energy intake, but the need for specific micronutrients means that foods are not always comparable based on energy content. Although sodium is essential for many physiological functions, many organisms face challenges obtaining it, especially those relying on a plant-based diet. Flowering plants often lure pollinators by manipulating nectar carbohydrate content, but nectar sodium might also influence pollinator choice. We experimentally tested how sodium addition and nectar carbohydrate concentration interact to affect bumble bee foraging decisions by allowing bees to forage on artificial flowers containing either low- or high-sucrose nectar, with or without sodium. We also explored whether sugar and sodium content in floral nectar trade-off across plant species. Bees preferred sodium-enriched nectar and preference nearly doubled when sucrose concentration was low. Furthermore, bees switched more often from flowers without sodium to those with sodium when sucrose concentration was low. Given increased fidelity for artificial flowers with nectar sodium, sodium may enhance plant fitness by increasing conspecific pollen receipt. Yet despite behavioral evidence that sodium enhances the perceived quality of low-sucrose nectar for bees, we found no evidence for a nectar sugar-sodium trade-off across plant species. We discuss implications for plant-pollinator interactions and flower evolution.

Foraging hematophagous flies such as the stable fly, Stomoxys calcitrans, are shown or expected to sense multiple characteristics of visual host cues. These include relative intensity, spectral composition, and the angle and degree of polarization. Here, we investigated the role of each of these visual cue characteristics on stable fly attraction. In laboratory two-choice bioassays, we offered groups of female or male stable flies a choice between paired photostimuli that differed in their visual characteristics, recording the flies' landing responses on stimuli as the response criterion. Bioassay flies preferentially selected lower-intensity visual stimuli even when paired stimuli markedly differed in spectral composition and polarization characteristics (a high degree of horizontal or vertical polarization). When paired stimuli had identical relative intensity, flies failed to discriminate between the stimulus that presented ultraviolet, blue, green, and red wavebands and a corresponding paired stimulus lacking one of these wavebands. Our results support the hypothesis that foraging stable flies may respond to colour and polarization characteristics of visual stimuli as modulations of perceived intensity. Acquired knowledge on the mechanisms that underlie stable fly visual attraction may inform the design of vision-based traps so that they become optimally attractive to stable flies and other biting flies.

Despite being a topic of early experimental studies, the effects of immersion-emersion cycles and temperature on the heart rate of intertidal Mytilus mussels have not been fully characterized. Using a recently released intertidal chamber paired with our own software package, we conducted a multiday experiment and continuously monitored the heart rate of the blue mussel Mytilus edulis. Under normal conditions, we detected a subtle spike in heart rate upon emersion before the eventual decline, as well as an overshoot upon reimmersion. Additionally, heart rate was sensitive to temperature changes during immersion but less responsive during emersion. These patterns were markedly altered following an unintended prolonged emersion event, which led to the loss of the emersion spike, an extended reimmersion overshoot, and heightened thermal sensitivity on top of an overall elevation in heart rate. However, the heart rate gradually returned to the pre-event baseline over the following days, demonstrating the significant resilience of the mussels. Our study highlights the potential of high-resolution physiological data in assessing the effects of environmental stressors on coastal ecosystems.

Abstract The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) controls behaviours that are essential for the parasitic lifestyle of plant parasitic nematodes (PPNs). Here, we used serotonin immunochemistry and in situ hybridization for the serotonin synthesis genes, Gpa-tph-1 and Gpa-bas-1, to map its signalling pathways in Globodera pallida J2 infective stages. We identified fourteen serotonin-immunoreactive cell bodies around the pharyngeal median bulb and revealed that eleven of them co-express Gpa-tph-1 and Gpa-bas-1, confirming these are serotonin-synthesising neurons. Serotonin immunoreactivity was also localised in structures consistent with labelling of amphids, and in sixteen non-neuronal cells located in the middle and posterior region of the nematode. Depletion of serotonin with reserpine reduced the serotonin immunoreactivity in all fourteen cell bodies confirming their neuronal identity, but not in non-neuronal tissue. We identified additional, but infrequent expression of Gpa-tph-1 in two cells and expression of Gpa-bas-1 in one pair of symmetrically bilateral cells anterior to the median bulb, that lacked serotonin immunoreactivity. The results reveal distinct pools of serotonin distribution in G. pallida and indicate serotonin signalling is more complex than in other parasitic nematodes and C. elegans, highlighting evolutionary divergence in neurotransmitter identity.

The spatial visual acuity of an eye is determined by both the spatial sampling density of the retina and the angular sensitivity of individual photoreceptors. In insect compound eyes, these are respectively characterised by the interommatidial angle, {Delta}{varphi}, and photoreceptor acceptance angle, {Delta}{rho}. During movement, however, finite photoreceptor integration times cause retinal image motion to broaden the effective acceptance angle, potentially degrading spatial resolution beyond its anatomical limit. This problem is especially acute in flying insects, which experience large rotational velocities during locomotion and must actively stabilise gaze to limit retinal image motion. Butterflies present a particularly demanding case because their flight is characterised by large, wingbeat-coupled thoracic oscillations. Here, we quantify how gaze stabilisation limits motion blur in freely flying Monarch butterflies Danaus plexippus by combining free-flight kinematic reconstruction of the head and thorax with separate intracellular measurements of photoreceptor acceptance angles and integration times from quiescent butterflies. We found that, without head compensation, thoracic oscillations would broaden the effective photoreceptor acceptance angle from its quiescent value of 2.17{degrees} to 9.38{degrees} during take-off. Compensatory head movements substantially reduced this motion-induced broadening, limiting the effective acceptance angle to 5.28{degrees}. Nevertheless, this 2.4-fold broadening of effective photoreceptor acceptance angle represents a substantial degradation of spatial acuity in flight, highlighting motion blur as an important constraint on the visual ecology of flying butterflies.

Undersea and high-altitude operations regularly put operators at risk for decompression sickness (DCS), which is attributed to bubble formation and growth during decompression. DCS mitigation strategies are validated with empirical testing, often conducted in hyperbaric chambers, which provide precise control of the pressure-time profile. A degree of variability for study outcome is anticipated in both decompression sickness (DCS) and venous gas emboli (VGE) endpoints. Experimentation in a chamber offers precision, but is not always practical or operationally relevant, leading researchers to use open water for the dive exposure. Dive profile consistency may vary by diver skill level and environmental conditions, potentially introducing dive profile deviations, which may be a factor of individual variability. There does not exist a quantitative framework for evaluating dive profile consistency and quantifying deviations in open water studies. In this work, we evaluate existing dive profile metrics and propose a workflow to estimate dive profile deviations using the Buhlmann dissolved gas phase model. The dive profile metrics included depth and time-based calculations, as well as surface gradient factor, degree-of-conservatism and the root mean square error of the Buhlmann tissue compartments. We developed a set of simulated dive profiles and obtained a set of 40 open water dives previously collected by certified divers, both of which were used to evaluate performance of the dive profile metrics. Depth and time-based metrics offered sensitivity to varying depths and time durations of the dive profiles, respectively. For the field dives, we found the Buhlmann root mean square error metric performed best at identifying the deviation profile, followed by pressure root time. These evaluations demonstrate the utility and limitations of previously published dive profile metrics and emphasize the importance of accounting for dive profile consistency in open water decompression trials.

Many animals can sense the Earth's magnetic field and use it for orientation. However, the mechanism underlying magnetoreception has not been identified, partially due to the lack of reproducible and genetically tractable model systems. Here, we establish a directional assay for planaria to test their responses to different magnetic fields. Girardia dorotocephala showed a preferred magnetic directionality in the horizontal plane in an Earth-strength magnetic field in the absence of visible light, which shifted if the azimuth was rotated and was absent when the magnetic field was cancelled. This directional preference differed between labs. Furthermore, the directional response was affected by a short but strong magnetic pulse, resulting in a random distribution. The effect of the pulse was temporary, with directional preferences recovering over several days. Taken together, these data are consistent with a magnetoreception mechanism in planarians that functions independently of light and relies on permanently magnetised material, such as single-domain magnetite. As planarians are genetically tractable and well-established, they are a promising invertebrate model organism that might offer insights into the mechanisms underlying magnetoreception in animals.

Crickets are gaining attention as sustainable protein sources because of their high nutritional value and low environmental impact. Growth models are essential tools for optimising production by predicting growth characteristics. However, previous studies have not compared multiple non-linear growth models for crickets or explicitly considered environmental factors. We analysed the growth patterns of the two-spotted cricket (Gryllus bimaculatus) and the Asian cricket (Teleogryllus occipitalis) at multiple temperatures (25.0, 27.5, 30.0, 32.5 and 35.0 {degrees}C) using three non-linear models (Gompertz model, logistic model, and von Bertalanffy model) and combined the predicted body mass with observed survival rates to predict the optimal harvest time and temperature. All three models fitted the data well (R2 [≥] 0.97 in both species). The asymptotic body mass was highest at 30.0 {degrees}C, and the growth rate constant increased with temperature, reaching its maximum at 35.0 {degrees}C except for the logistic model in G. bimaculatus (32.5 {degrees}C). In addition, the predicted value of harvest, calculated by combining predicted body mass and observed survival probability, was highest at 30.0 {degrees}C in both species, reaching 13.82 kg/m3 at 38 days in G. bimaculatus and 7.12 kg/m3 at 62 days in T. occipitalis. These results suggest that 30.0 {degrees}C is the most suitable constant-temperature condition for maximising predicted productivity, although the optimal harvest timing differs between species.

Tardigrades, key ecdysozoan organisms, have poorly characterized daily behavioral rhythms linked to their molting (ecdysial) cycle. We continuously tracked the locomotor activity of the model tardigrade Hypsibius exemplaris using infrared videography and deep learning-based automated tracking under three photic regimes: a light/dark cycle, constant darkness following entrainment, and constant darkness without prior entrainment. We identified a robust endogenous circadian-like rhythm in locomotor activity that persisted in constant darkness, independent of photic input. Although the light/dark cycle enhanced the rhythm amplitude and the overall animal activity, it was not required for rhythmicity. Individual phases varied widely, indicating a lack of population-level synchronization. Furthermore, we developed a behavioral classification system based on activity level, which enables categorizing distinct behaviors. Behavioral patterns were modulated by distinct ecdysial stages, with quiescence gating premolt and time-of-day dependent patterns in feeding and wandering. These findings provide evidence consistent with an endogenous, circadian-like timing system in tardigrades that integrates daily activity with molting cycles and establish H. exemplaris as a promising model for studying ecdysozoan chronobiology.

From the iconic V-formations of geese to the spectacular murmurations of starlings and communal roosting of millions of bramblings, bird flocks keep sparking wonder and scientific curiosity. In science, common themes include how flocking formation impacts the energy expenditure during migration, how it affects risks of predation, how it relates to information spread on food abundance, and how birds manoeuvre in dense flocks. Here, we report on observations of a very different type of flock formation: migrating terns over open sea flying side by side in a search party manner -- strikingly orthogonal to their heading -- with interbird distances up towards 300 m and flocks of around ten birds covering widths up to one kilometer. Such sparse formations of migrating birds stand in great contrast to the classes of bird flocks previously described in the literature. We present simultaneous field observations and ship-based radar measurements of flock characteristics and argue that this is a form of fly-and-forage migration that may be a common, but overlooked, network foraging strategy of surface feeding birds: while most migrants cannot combine migration and foraging, the situation is different for surface feeders like terns.

Most animals move intermittently. Of these, animals that fly and swim move in two stages: during active propulsion, when they beat their wings or swing their tails, and right after, when they glide or coast through the air or water. The kinematics of active movements are typically well studied, but the coasting period afterwards is less understood. We used larval zebrafish as a model of intermittent locomotion. Larval zebrafish swim in discrete bouts lasting ~200 ms, coast for short durations and distances, and then are largely stationary for ~800 ms. We find that larval zebrafish do not coast like a passive object. While they coast for distances that are correlated with the prior swim's strength, as expected from physics, they seem to be able to modulate the mechanics of their coasting: the time constant is not a constant, but varies from coast-to-coast. Furthermore, the time constant is correlated with the duration of the interval between swim bouts. This suggests that larval zebrafish can either shorten or extend their coasting duration, and that coasting inhibits the initiation of the next swim. Finally, the time constant changes quickly, while other swimming kinematics (e.g., distance swum) are autocorrelated over the course of at least several seconds. Altogether, this suggests that fish, and perhaps other animals that move intermittently, actively modulate their post-propulsion movement, possibly to gather sensory feedback in the absence of blurry visual feedback associated with fast locomotion.

Many animals use the Earth's magnetic field to orient and navigate over long distances, yet the sensory mechanism that underlies this ability remains unresolved. A central question is whether the compass depends on light: a cryptochrome-based radical-pair mechanism, best characterised in birds, requires a photon to generate the magnetically sensitive species, whereas magnetic particle-based and inductive mechanisms do not. In mammals, which lack the light-sensitive cryptochromes that the avian model requires, this question is unresolved. Here we tested whether migratory bats, Pipistrellus nathusii, require light for migratory orientation, studying wild migrants at a major flyway under laboratory-controlled light conditions, including verified total darkness. Bats were acclimated to starlight-level illumination or to total darkness before release into a circular arena in which take-off direction was recorded, and take-off latency was taken in every trial as a control for behavioural state. Bats released under starlight-level illumination, and bats released in total darkness after starlight acclimation, both oriented in the south-westerly direction typical of free-flying bats at this flyway; their mean headings lay 11 degrees apart and did not differ in direction, in angular concentration, or in take-off latency. Bats released in total darkness after prolonged darkness produced the same mean heading but did not reach significance under a Rayleigh test, and did not differ from either other group. To our knowledge, this is the first demonstration that a migratory mammal expresses its seasonally appropriate migratory heading in total darkness. Bats orienting in darkness took off five to eight minutes after their last photon exposure, seven to eight orders of magnitude beyond the microsecond lifetime of the only cryptochrome radical pair demonstrated to exist and to be magnetically sensitive. Because a compass reading is informative only at the moment of the directional decision, a signal generated before a bat entered darkness could not encode its heading at take-off; the magnetically sensitive species must therefore form without photons. The radical pairs capable of doing so have not been shown to exist, even in vitro. A migratory mammal thus expresses its seasonal heading without concurrent light, in a lineage lacking the photoreceptor on which the light-dependent compass in birds relies.

Ambient illumination influences many aspects of brain function, including responses to threats. One brain region proposed to mediate these effects is the periaqueductal gray (PAG), an evolutionarily conserved midbrain structure that contains multiple subpopulations of peptidergic neurons. Here we ask which cell types in the larval zebrafish PAG respond to changes in ambient illumination. By registering post hoc gene expression to neurons recorded during exposure to sensory stimuli, we show that GABAergic PAG neurons expressing the neuropeptide Relaxin-3 (rln3a+) are persistently activated by darkness, while a neighboring subpopulation of GABAergic PAG neurons expressing penka is not. Zebrafish lacking rln3a show a reduction in locomotion during darkness. We propose that darkness recruits Relaxin-3-releasing PAG neurons to promote vigilance and potentiate defense circuits. These results identify a specific peptidergic population of PAG neurons that are potentially relevant to behavioral state dysregulation associated with excessive darkness.

Animal pose estimation typically requires manual annotation of body parts to train neural networks for each new recording system or environmental condition. Here we use a general-purpose vision-language model (VLM) to generate annotations in multi-animal recordings of fruit flies (Drosophila melanogaster). Initial annotations follow written anatomical descriptions and are reviewed by an independent VLM session using neighboring video frames. On 120 held-out fly images, AI annotations had a median distance of 2.28 pixels from human labels, with 84.0% agreement within eight pixels, averaged across landmark types. The comparison included 10 landmarks, with head and eyes analyzed separately because of systematic differences in placement. Review improved agreement primarily at the feet, with the largest remaining discrepancies at positions with limited visual evidence. Visual inspection revealed cases in which the AI annotations were more anatomically accurate than those of an experienced human annotator, particularly at the feet. A convolutional network trained using SLEAP on 300 AI-annotated fly images showed similar agreement with human labels and closely reproduced the AI annotations on held-out images, with a median distance of 1.56 pixels. We also applied the procedure to recordings from another laboratory, using new AI annotations to train a separate pose network. Although VLM annotation required minutes per fly, the trained networks enabled pose estimation throughout more than 700,000 frames. These results demonstrate that VLM annotations can support fast animal pose estimation without manually labeled training images.

Animals navigate through dynamic environments in search of resources. Dopaminergic systems are known to control movement and assign motivational value to cues, actions and outcomes, but how they guide ongoing locomotion towards a goal remains unclear. Here, we demonstrate that Drosophila mushroom body innervating dopaminergic neurons encode steering signals in world-centred (allocentric) and body-centred (egocentric) coordinates during goal-directed navigation. Thirsty flies track water vapour through upwind directed turning and locomotion, that persists for several seconds after the vapour disappears. We identified a population of rewarding dopaminergic neurons to be specifically required for maintaining directed tracking following vapour offset. Recordings of these dopaminergic neurons during behaviour revealed delayed bilateral activity that followed upwind-directed turning in allocentric coordinates. Moreover, lateralized ipsilateral activity in a subset of these dopaminergic neurons encode whether the fly turns left or right in egocentric coordinates. Finally, these dopaminergic neurons also exhibited increased ipsilateral activity when turning in the direction where water vapour was previously encountered. Therefore, activity of mushroom body dopaminergic neurons represents steering and direction-specific actions that together maintain goal-directed tracking in turbulent environments.