Journal of Experimental Biology

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.

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.

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.

Developing in suboptimal temperatures can have widespread negative effects in organisms, but early exposure to thermal challenges can also trigger increased investment in thermoregulatory capacity. Within birds, the effects of cold exposure during incubation may differ between precocial and altricial species due to differences in the ontogeny of thermoregulation. To date, most of the evidence showing that embryonic cold exposure shapes future thermoregulatory capacity comes from a few precocial species in laboratory settings. Less is known about sensitivity to low-temperature incubation in altricial species which only become fully endothermic after hatching. In this study, we tested the effects of cold temperatures during embryonic development by temporarily cooling the nest boxes of free-living tree swallows (Tachycineta bicolor) during late incubation and subsequently exposing nestlings to an acute cold challenge. Cold exposure during early development made nestlings better at maintaining their body temperatures during the acute challenge. Developmentally cold-exposed birds also had higher thyroxine (T4) levels when sampled at lower ambient temperatures, whereas control birds showed the opposite relationship. Among nestlings that failed to maintain their body temperature during the acute cold challenge, developmentally cold-exposed nestlings secreted more corticosterone during the acute cold challenge than control nestlings. Despite these changes, cold-exposed birds did not differ from controls in cold-induced metabolic rates, body mass, or bacteria killing ability. These results are, to our knowledge, the first to show that developmental cold exposure increases the capacity to maintain body temperature during future cold challenges in a songbird. These findings suggest that experiencing even a mild decline in ambient temperatures during embryonic development could prime birds to cope more effectively with harsh or variable environmental conditions later in life.

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.

Multifunctional anatomical structures must accommodate potentially competing performance demands, leading to functional tradeoffs. The mammal rostrum (snout) is involved in multiple functions, including feeding, respiratory conditioning, olfaction, and, in most bats, echolocation. Therefore, it serves as an ideal system to investigate whether and how functional tradeoffs promote or constrain morphological diversification. While the external morphology of the rostrum has been linked with mechanical demands of feeding in bats, the factors and processes influencing its internal morphology, including the turbinals in the nasal cavity, remain comparatively unexplored. Here we investigate how internal rostral morphology reflects specialization and tradeoffs among respiratory air conditioning, echolocation, and olfaction by quantifying turbinal morphological diversity in a phylogenetically and ecologically diverse sample of bat species. We find that echolocation mode predominantly influences both the ethmoturbinal complex (ETc) and maxilloturbinal (MT) systems, with nasal-emitting bats having reduced and less complex turbinals compared to oral and non-laryngeally echolocating bat species. MT complexity and surface area are not associated with bioclimatic variables representing thermal variability and aridity. However, the ETc exhibits higher complexity in plantivorous bats than faunivorous bats, suggesting higher olfactory reliance. While MT morphological disparity was predominantly explained by echolocation mode, ETc morphological disparity was associated with both echolocation mode and diet, suggesting potentially opposing demands favoring turbinal reduction for sound transmission and elaboration for olfaction. Among nasal emitters, which must accommodate both demands within the nasal cavity, ETc complexity exhibited higher relative disparity compared to MT complexity. These patterns suggest that competing functional demands may promote or maintain morphological diversity, possibly through ecological specialization and/or many-to-one mapping between morphology and performance.

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.

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.

The most studied symbiosis between land vertebrates and algae, is the one between Ambystoma maculatum and Oophila amblystomatis, where the latter colonizes the amphibian''s eggs. Although clear benefit given by algal growth for the salamander embryo has been observed, mutualism is not widely accepted. Mutualistic hypotheses posit benefits from the host to the algae which include protection, N- and C-sources, but no experimental evidence has been obtained. In fact, recent studies suggest that the algae are stressed during this relationship and propose fermentation rather than photosynthesis as main driver for algal growth. In this work, we analyzed embryonic host developmental rate, algal and embryo growth and photosynthetic function of eggs housed at different light intensities. We provide evidence that photosynthetic growth is the main driver for Oophila and that higher algal growth yields faster developmental rate and embryo size. Also, we provide strong evidence that rates of photosynthetic electron transport (and the concomitant O2 evolution) must be higher than those of consumption by the embryo for this symbiosis to be mutualistic. Since photosynthetic rate is dependent on light, this work illustrates the importance of light intensity in this symbiotic relationship.

Known for their rapid water surface locomotion, whirligig beetles (Gyrinidae) are also fast divers. They can transition from the surface to underwater swimming in under 100 ms. Diving requires them to generate sufficient hydrodynamic torque to break the water surface and reorient the body. However, the mechanism by which whirligigs create the necessary pitch-down torque is unknown. Here, we show that their diving is achieved by bending the abdomen. The inertial hydrodynamic pressure on the flexed abdomen generates the torque necessary for surface breaking and body rotation. The torque is maximized at an abdominal angle of 45{degrees}, whereas for effective diving, whirligigs display a preferential abdominal angle of around 28{degrees}, closely aligning with the angle that maximizes the torque-to-drag ratio. This abdominal angle is optimized by balancing torque generation with the drag penalty. Our findings inspire a mechanism for surface-to-underwater transition, offering design principles for multimodal robots capable of locomotion across the air-water interface.

Precocious reproduction, prior to the acquisition of the final adult morphology, occurs in a variety of animals and represents a distinct life-history strategy with important consequences for growth and survival. Larval reproduction is far rarer and usually restricted to groups that go through metamorphosis. In ctenophores, Carl Chun described dissogeny more than 130 years ago, a distinctive life-history pattern involving larval and adult reproductive periods separated by gonadal regression. Yet, the potential plasticity of larval reproduction in ctenophores remains unresolved. Here, we developed a method to follow and quantify reproductive output in Mnemiopsis leidyi larvae and tested environmental factors that may regulate precocious reproduction. We found that conspecific density strongly influences its onset: low-density, well-fed animals can bypass reproduction at the cydippid stage and develop rapidly into the lobed form, whereas higher density promotes precocious reproduction. Food availability and associated growth rate also contribute but cannot alone explain the observed differences. Once reproduction is established, however, high conspecific density suppresses reproductive output, an effect reproduced by exposure to seawater incubated with high M. leidyi densities, consistent with water-borne chemical communication. Salinity and light conditions further modulate spawning. Together, our results reveal substantial environmental plasticity in precocious reproduction in M. leidyi, reconcile long-standing contradictory observations of ctenophore development, and highlight how environmental cues can reshape reproductive life-history transitions.

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.

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.

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.

Heat-induced sterility is widespread across ectotherm species, yet its underlying mechanisms remain poorly understood. In mated females, thermal stress can disrupt female reproductive physiology while also affecting sperm stored within the reproductive tract, making the mechanisms of fertility loss difficult to distinguish. Here, we investigated the extent and recovery dynamics of female heat-induced fertility loss across seven Drosophila species. We tracked fertility after acute heat exposure, tested whether remating restored fertility, quantified stored sperm in two closely related species, and examined whether reproductive thermal sensitivity covaried with adult heat tolerance. We found that heat exposure reduced egg-hatching success in all seven species, but recovery differed among species. Fertility recovered within several days in most species, whereas D. simulans showed persistent declines and D. melanogaster showed limited recovery. Remating restored or maintained fertility in both species. Stored sperm declined through time in both D. simulans and D. mauritiana, but D. mauritiana retained more sperm at later sampling points, consistent with their contrasting patterns of fertility decline and maintenance. Across species, greater adult heat tolerance was associated with smaller immediate reductions in fertility, but not with cumulative reproductive loss across subsequent days. Together, these findings show that similar initial fertility losses can lead to markedly different recovery trajectories and highlight the importance of comparative, multi-species approaches that integrate female physiology, sperm-storage dynamics, mating behavior, and whole-organism thermal tolerance when assessing reproductive resilience to climate warming.

Synchrony between time-of-day and food intake is critical for maintaining homeostasis while desynchrony between these cues caused by repeated bouts of mistimed eating (ME) is profoundly disruptive to organismal health. However, it is unknown whether the same is true for a single bout of ME. While it is generally accepted that mammals recover quickly from acute alterations in feeding schedule and that chrono-disruption requires chronic, repeated changes, there have been no dedicated investigations of acute ME to suggest otherwise. Therefore, we exposed female and male C57Bl6/J mice to a single bout of ME or timed eating (TE) and continuously measured metabolic and behavioral responses for five days. A single bout of ME was sufficient to disrupt the daily rhythms of metabolic and behavioral outcomes for several days. Energy expenditure and wheel running activity were disrupted and light:dark variation in food intake and macronutrient utilization were altered for several days. Finally, ME changed the amount of time spent resting in for several days post-ME. The TE paradigm disrupted macronutrient utilization for two days but otherwise had no effect on metabolism or behavior. Our results demonstrate that one instance of ME alone has lasting impacts on daily rhythmicity and is an understudied source of circadian disruption.

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.

Marine heatwaves cause widespread coral bleaching and mortality, yet thermal responses often differ between adult and juvenile corals, with the latter frequently being more tolerant. Whether the observed enhanced tolerance of juveniles reflects intrinsic physiological differences across life stages or simply reduced environmental stress due to shaded microhabitats occupied by juveniles, or a combination of both factors, remains unclear. To test whether colony size determines thermal responses and if a threshold size exists for tolerance, we exposed 89 colonies of the reef-building coral Acropora aff. digitifera to a 6-week, 32.5 C simulated marine heatwave under controlled light conditions, with cumulative stress reaching 20 C-weeks. Contrary to expectations, ~2 C-weeks less heat stress was required to trigger bleaching and mortality in coral juveniles compared to adults. Mortality divergence across colony sizes was greatest at ~10 C-weeks, when the smallest corals exceeded 80% mortality, whereas the largest colonies showed <10% mortality. A breakpoint in survival odds was apparent at 13.4 cm diameter, corresponding to the size at onset of sexual maturity in the native wild population for this species. These results indicate an ontogenetic shift in physiological heat tolerance, with larger reproductively mature colonies showing enhanced tolerance. The apparent survival advantage of juveniles in the reef frequently reported during bleaching events could be more likely due to micro-environmental buffering than intrinsic physiological tolerance. Our findings suggest that juvenile corals may be more vulnerable to climate change than previously thought, particularly as declining habitat complexity could reduce microhabitat refugia on future reefs.

Crank length affects the geometry of the bicycle-rider system and influences the motion of the lower limb throughout the pedal cycle. Conventional crank selection has largely relied on rider stature and mechanical power, yet mechanical power and cycling economy remain remarkably insensitive to crank length across a broad range. Yet, elite cyclists and triathletes increasingly adopt shorter cranks, particularly in disciplines where aerodynamic drag strongly affects cycling performance. However, the mechanisms that connect crank length to mechanical power and aerodynamic drag remain unclear. Here we show that crank length couples rider anthropometry, hip kinematics, and aerodynamic drag. Population anthropometry of 6,068 individuals reveals that stature alone poorly predicts personalized crank length, while geometric analysis identifies a nearly linear relationship between crank length and minimum lower hip angle: a 10-mm reduction in crank length increases hip-angle margin by approximately 1.42 degrees with little mechanical penalty. This geometric freedom permits more flexion of the torso and a more aerodynamic posture; the resulting aerodynamic advantage grows rapidly with cycling speed because aerodynamic power scales with velocity cubed. Data from 150 elite cyclists and triathletes support this shift toward shorter cranks, with triathletes adopting shorter relative crank lengths than cyclists. Our results establish crank length as a personalized design variable that connects lower-limb geometry to aerodynamics. These insights enable personalized crank selection, integrated rider-bicycle design, and performance optimization for the increased aerodynamic demands of high-speed cycling.

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.