The Journal of Experimental Biology

Octopuses exhibit remarkable regenerative capacity, as shown by their ability to fully restore a whole arm. During this process, a complex appendage containing muscle, connective tissue, vasculature, suckers, sensory structures, peripheral nerves and the axial nerve cord (ANC) is regrown. Although the morphology of octopus regeneration is increasingly well described, the physiological variables that determine regenerative speed, including the potential contribution of ion-channel-dependent processes, remain poorly understood. In this work we examined the influence of reproductive state, injury severity, biological sex and magnesium chloride (MgCl2) anesthesia, on the rate of longitudinal arm regrowth. Reproductive stage was the strongest determinant of regenerative performance: pre-reproductive stage (pre-RS) animals showed robust regrowth, whereas reproductive stage (RS) animals regenerated significantly slower. In pre-RS animals, regeneration speed scaled strongly with injury severity, whereas this relationship was absent in RS animals. Biological sex did not independently predict regeneration speed, and MgCl2 exposure showed no long-term inhibitory effect on regeneration. Our final model explained 81.4% of variance in regeneration speed, with reproductive state and injury severity having major effects. These findings demonstrate that sexual maturation and optic-gland-mediated senescence act as an irreversible physiological block against somatic regeneration, and provide empirical support for mechanosensory and bioelectric-dependent scaling models of cephalopod limb restoration.

Evolutionary patterns of regeneration ability in animals may reflect the relative costs of injury versus regeneration on organismal function. While the cost of injury seems obvious, the investment of resources and other physiological effects produced by regeneration may be considerable, and the effects of both injury and regeneration may depend upon myriad factors intrinsic to organisms or within their environments. Such effects and how they vary are not well understood. We investigated the physiological effects of amputation injury and subsequent regeneration of tissue in the annelid Pristina leidyi, using environmental stress tolerance as a relevant measure of performance. Unexpectedly, injury improved survival under heat stress, but improved heat tolerance was unrelated to changes in metabolic rate. We performed TagSeq to investigate the transcriptional basis of injury-induced heat tolerance and found that injured worms produced an exaggerated response to subsequent heat stress compared to either injury or heat stress alone. Both injury and heat stress commonly induced the expression of just two heat shock family proteins. Pharmacological inhibition of one of these proteins, mortalin, drastically reduced heat tolerance in both uninjured and injured worms and substantially impaired anterior tissue regeneration. We posit that injury-induced expression of mortalin and other cellular stress response factors briefly confers improved resistance to broad forms of stress. Our work demonstrates that injury may have unpredictable effects and points to a shared ancestral response to various forms of biological damage with a critical role in animal function during the regeneration process.

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.

Background: Insufficient oxygen (hypoxia) or complete lack of oxygen (anoxia) causes cellular dysfunction and death in terrestrial animals. As a result, animals have developed homeostatic strategies, including changes in movement strategies to escape and survive stressful environments. Caenorhabditis elegans respond to changes in oxygen by increasing the frequency of reversals (backward movement) and increasing locomotor speed; these behaviors enable them to escape unfavorable environments and move to more suitable ones. The molecular and neural mechanisms underlying behavioral responses to low-oxygen environments are incomplete. Neuropeptides regulate C. elegans behaviors by modifying neural circuit function in response to hypoxia. Loss of function in C. elegans neuropeptide nlp-40 and its receptor aex-2 increased survival to anoxia [1]. However, the cells in which NLP-40 and AEX-2 function for anoxia, as well as the mechanisms underlying responses to anoxia, remain elusive. We hypothesized that NLP-40 and AEX-2 regulate anoxia by altering escape behaviors. Results: We found that neuropeptide NLP-40 functions in cholinergic neurons and AEX-2 functions in cholinergic or GLR-1 interneurons or motor neurons to regulate susceptibility to anoxia. We found that loss of either nlp-40 or aex-2 decreased locomotor activity and reversal frequency early during 48 hours of anoxia, suggesting that nlp-40 and aex-2 are required for the proper execution of these behaviors. We found that neuronal knockdown of nlp-40 recapitulated the behaviors observed in nlp-40 mutant animals and increased survival; however, restoring aex-2 function in glr-1 interneurons or motor neurons was sufficient to rescue behaviors to wild-type and restore vulnerability to anoxia. Conclusions: Overall, our results suggest that NLP-40 and AEX-2 likely function in cholinergic or glr-1 interneurons or motor neurons to regulate escape behaviors during anoxia. Escape behaviors are part of the homeostatic response and are typically protective in the wild; however, in our paradigm, we show that loss of either nlp-40 or aex-2 reduces escape responses and confers protection against 48 hours of anoxia. Collectively, these results highlight a new role for NLP-40 and AEX-2 in anoxia and shed light on mechanisms underlying the adaptive responses to anoxia.

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.

Simulated microgravity provides a controlled perturbation for testing how loss of mechanical load affects human tissues. After 24 hours in rotating-wall vessels, neural, cardiac and tonsil organoids developed tissue-specific transcriptional responses that overlapped published spaceflight datasets, accompanied by higher transcriptomic-age estimates in neural and cardiac models, altered cardiac-organoid contraction dynamics and modified tonsil-organoid antiviral gene-expression signatures. These findings establish a multi-organoid framework for studying acute responses to gravitational unloading.

Oxygen is essential for all terrestrial animals, but there is dramatic variability in how well different animals and even different cell types can adapt to reduced oxygen availability. We used a meta-analysis of the literature, with a focus on mouse studies, to identify pathways that might act to protect animals in low oxygen environments. We identified 108 genes whose mRNA levels change under hypoxia, and 55 genes critical for mounting a response to hypoxia. With this data, we developed a list of conserved genes, and we tested three C.elegans genes previously uncharacterized in hypoxia, mxl-3, yap-1, and ador-1, and found that loss of function altered egg-laying during and after hypoxia. Our method provides a more targeted approach of how to screen for hypoxic phenotypes and study in more genetically tractable organisms to show mechanisms.

Extraocular motoneurons are the final neuronal relay implicated in gaze motor control and are known to be subdivided in functional subgroups, differently implicated in ocular motion dynamics. However, the maturation of these functional populations of extraocular motoneurons, in relation with the development of gaze-stabilizing reflexes remains largely unexplored. In amphibian tadpoles, the angular vestibulo-ocular reflex (VOR) appears later than other visuo-vestibular ocular reflexes and matures until the metamorphosis climax. Two types of Abducens motoneurons have been described to participate to the angular VOR in larval frog: spontaneous motor units, exhibiting a robust resting activity and silent motor units recruited only during head motion. The aim of this study was to investigate the maturation of these two types of Abducens motor units in relation with the development of the angular VOR by evaluating their discharge dynamic in response to head rotation in semi-intact preparations of larval Xenopus laevis. During larval life, the discharge modulation during sinusoidal head rotations increases significantly for silent units only, demonstrating a better sensitivity of this Abducens motoneuron sub-population to horizontal semicircular canal activation. In addition, this functional maturation was accompanied by an increase of the myelination in the lateral rectus motor nerve, promoting a faster conductivity in late larval stages than in early one. These findings showed that the development of the angular VOR is supported by a selective maturation of extraocular motoneurons subpopulations, specifically implicated in the improvement of the ocular kinematic during the reflex.

Ankle stiffness is decreased during movement compared to posture; however, the etiology of this decrease remains unknown. Determining what gives rise to this decrease is critical for understanding how humans successfully interact with their physical world and how that ability is compromised by functional impairments. While the triceps surae and Achilles tendon primarily dictate ankle stiffness, the relative contributions across posture and movement remain unknown. Therefore, our study sought to quantify the relative contributions of the muscle and tendon to ankle stiffness and how those contributions differ between posture and movement. We used our technique, which combines B-mode ultrasound imaging with joint-level perturbations, to quantify ankle, muscle, and tendon stiffness simultaneously. Since ankle, muscle, and tendon stiffness all scale with torque, participants matched torque between posture and movement tasks. During posture, the Achilles tendon is the dominant contributor to ankle stiffness. However, during movement, the triceps surae and Achilles tendon contribute more equally to ankle stiffness, which can be attributed to a significant decrease in muscle stiffness during movement. Here, we provide the first empirical data on how state-dependent properties of the triceps surae and Achilles tendon contribute to ankle stiffness in conditions relevant to locomotion.

In most muscles, contraction is initiated by neural activation. Some groups of insects break this rule, flapping at frequencies far exceeding the neural drive to their flight muscles. These insects muscles (termed asynchronous) produce force in response to stretch, enabling flight at faster frequencies than would be possible through the slow calcium-dependent processes associated with neural activation. The first flapping insects lacked stretch-activated physiology, which then evolved on top of neural activation dynamics before likely being reduced again in some groups including moths. Stretch and neural activation can co-exist, but it remains unclear if stretch-activation alone is sufficient to generate asynchronous flapping in insect flight muscle. Building on prior closed-loop muscle physiology platforms, we develop a new way to perform a gain-of-function muscle physiology experiment called the physiological dynamic clamp. Inspired by dynamic clamp experiments in neuroscience, we couple isolated intact flight muscle from a hawkmoth, Manduca sexta, to simulated stretch-activation in virtual reality. Tuning virtual reality parameters allows us to manipulate the degree of stretch-activation in silico while retaining all other physiological properties of the muscle. With artificially enhanced stretch activation, we find that hawkmoth muscle can support stretch-activated work at typical wingbeat frequencies. When simultaneously stimulated at wingbeat frequency, interference between stretch and neural activation results in variable work production. However, this interference disappears when the two activation timescales are close to each other resulting in entrainment to the neural drive. Matching time scales suggests an evolutionary path for smoothly transitioning to stretch-activated, asynchronous flight and back again.

In many insects, females exhibit behavioral changes after mating. This plasticity can be mediated by interactions between male-derived seminal fluid proteins (SFPs) and the female nervous system. The Drosophila melanogaster seminal fluid protein Sex Peptide (SP) causes mated females to sleep less during the day and consume more food than unmated females, among other behavioral changes. Although the SP-sensing neural circuits that convey information to the females brain are known, the downstream neurons that affect post-mating behaviors remain unknown. P2 neurons in the flys central brain are good candidates for regulating post-mating changes in sleep and food consumption, as they express feeding- and sleep-responsive neuropeptide F (NPF), directly innervate the flys sleep center (the dorsal fan-shaped body, dFB), and are responsible for social-isolation-induced changes in sleep and food consumption. Using the GAL4/UAS system, we activated or silenced P2 neurons in mated and unmated females. We measured sleep duration using Drosophila Activity Monitors (DAM) and food consumption using Capillary Feeder (CAFE) and blue-dye assays. We found that P2-neurons are not responsible for post-mating changes in sleep and food consumption: mated females slept less during the day and ate more compared to unmated females irrespective of P2 neuron perturbation. Together, these results indicate that P2 neurons are unlikely to be primary downstream neurons through which male-transferred SP regulates post-mating sleep and food consumption. Future studies could determine whether P2 neurons regulate other post-mating behaviors or modulate sleep and feeding behaviors in a context-dependent manner.

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.

Environmental temperature strongly influences muscle contractile mechanics and locomotor performance in ectotherms, yet animals routinely develop across a range of temperatures while maintaining effective movement. We tested the hypothesis that developmental temperature induces compensatory changes in the intrinsic mechanical properties of the muscles that power the fast-start escape response in larval zebrafish (Danio rerio). Larvae were reared at 25{degrees}C, 28{degrees}C, or 32{degrees}C, and contractile properties of intact tail myotomal muscles were measured across experimental temperatures. Acute changes in experimental temperature strongly affected twitch kinetics, particularly relaxation rate (Q10 = 2.1), resulting in substantial changes in twitch duration. In contrast, rearing temperature produced adaptive changes that opposed these acute thermal effects. At a common experimental temperature, muscles from cold-reared larvae exhibited faster intrinsic relaxation and greater force production during shortening at a physiologically relevant velocity, whereas warm-reared larvae showed slower relaxation and reduced shortening force. As a result, twitch kinetics were largely normalized when measurements were made at each group's rearing temperature, reducing the apparent thermal sensitivity of relaxation rate (Q10 = 1.1). To identify molecular correlates of these functional adaptations, we performed label-free quantitative LCMS proteomic analysis. Cold rearing increased the abundance of Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) proteins, driven primarily by elevated atp2a1 expression, while warm rearing reduced the abundance of the major parvalbumin isoforms pvalb1 and pvalb2. These changes implicate remodeling of intracellular calcium handling as a mechanism underlying thermal compensation of muscle function. Together, our results demonstrate that developmental temperature modifies the intrinsic mechanical properties of larval zebrafish muscle in ways that counteract the direct effects of environmental temperature, thereby preserving the timing and power-generating capacity required for fast-start escape performance.

Neural circuits produce reliable activity even after environmental disturbances. This occurs because neurons respond to perturbations in a compensatory manner, a process termed homeostatic plasticity. Bullfrogs undergo prolonged periods underwater during winter, when lung ventilation and its neural control system ceases activity, but air-breathing resumes unscathed when environmental temperatures increase weeks to months later. Compensatory neural mechanisms that contribute involve upregulation of excitatory synaptic transmission on motoneurons driven by inactivity, but whether inactivity or acclimation to low temperatures drive other forms of compensation is not known. The GABAA receptor contribution to respiratory rhythm generation is downregulated following overwintering, which promotes network excitability. Therefore, we disentangled the contributions of cold temperature acclimation and inactivity experienced during overwintering on reduced GABAergic signaling. Here, we show that cold temperature, and not inactivity, reduces GABAA signaling in the respiratory rhythm generating network, without influencing GABAA transmission onto motoneurons. Therefore, cold temperature acclimation drives reduced GABAergic signaling selectively in inter-neuronal rhythm generating circuits, while excitatory motoneurons synapses are strengthened by inactivity in the overwintering environment. Most work interprets compensatory plasticity as activity-dependent during activity perturbations, but we reveal that different aspects of a disruptive environment elicit distinct forms of plasticity across a motor network.

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.

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.

To better understand how animals cope with increasingly variable and challenging environments, there is a need to study how prolonged exposure to elevated glucocorticoid hormones (i.e. one mediator of the stress response) affects their physiology. While glucocorticoid elevation is known to increase oxidative stress and accelerate cellular ageing, there is evidence that king penguins (Aptenodytes patagonicus) can prevent oxidative stress during acute stress exposure, suggesting that species may differ in their sensitivity to glucocorticoids downstream negative effects. As king penguins thrive in a seemingly harsh environment, we hypothesized that they may be able to limit the deleterious effects usually associated with chronic glucocorticoid elevation, either through resistance (i.e. prevention of downstream negative effect) or resilience (i.e. rapid recovery following transient negative effect). To test this hypothesis, we experimentally elevated corticosterone levels in incubating king penguins and quantified treatment effects on a suite of physiological traits at multiple time points across incubation and early chick-rearing, up to ca. 2 months after implantation. Corticosterone-treated individuals showed a prolonged increase in corticosterone and decrease in body condition, confirming our treatment likely mimicked sustained stress exposure. Heterophil-to-lymphocyte ratio was only increased transiently, and there was no clear evidence that treatment influenced oxidative stress or telomere length maintenance. Plasma energy metabolites were mainly affected early after implantation, with rapid recovery over time. Overall, our results suggest that adult king penguins show at least moderate resistance and resilience to chronic corticosterone elevation, especially in preventing cellular integrity loss, though at-sea physiological effects remain to be determined.

Many fish undergo dramatic, socially cued female-to-male sex change, yet the events that initiate and then regulate gonadal reprogramming and reorganisation during this phenomenal metamorphosis are broadly unknown. The New Zealand spotted wrasse, Notolabrus celidotus, is a temperate fish species that displays the extraordinary ability to undergo protogynous sex change. Removal of a terminal-phase male from a social group triggers the most dominant female to change sex and become a male. To characterise the molecular changes associated with gonadal metamorphosis in spotty, we used genomic and transcriptomic approaches, generating a high-quality genome and a transcriptomic time-series that captures the sex change process from start to finish. These data, together with paired histological data, reveal distinct transcriptional profiles that characterise the process of sex change. As expected, the expression of masculinising genes steadily increases, while feminising genes steadily decrease, throughout the transition. We further identify novel candidate genes, including genes involved in immune signalling and tissue remodelling through inflammation-mediated apoptosis, whose expression strongly correlates with key events in sex change, while also confirming the roles of known sex determination and differentiation genes in this process. Collectively these data give us new insights into how a normally committed developmental process remains plastic and is reversed to completely alter organ structures, and also sheds light on the evolution of sex determination in other animals. This work furthers the development of the spotty as a new tractable model for sex change research.

Brain microRNA (miRNA) expression can shift in response to the social environment, translating external cues into regulatory action that can influence brain structure, neural activity, and behavior. miRNAs are key drivers of behavioral flexibility, regulating gene networks involved in neuroplasticity. We manipulated social rearing conditions of brilliant-thighed poison frog tadpoles (Allobates femoralis) to test whether isolation-linked behavioral changes are associated with altered miRNA expression in the midbrain and eyes, tissues linked by the retinotectal tract, a known connection for trafficked precursor miRNAs. We asked whether isolation alters visually guided behavior using a light/dark preference assay and found that isolated tadpoles spent less of the trial on the dark side than did group-housed tadpoles. In a social place preference assay, group-housed tadpoles preferred a zone near a conspecific over an object control when provided with multimodal sensory access, but not when access was restricted to vision alone. We next tested whether social rearing conditions influenced miRNA expression and found that isolated tadpoles exhibited upregulation of miR-15-P1c_3p*, the canonically non-dominant arm of an understudied miRNA, in the midbrain and eyes. We detected a shift in arm dosing for miR-124, a miRNA with conserved roles in neural differentiation and plasticity. Finally, social isolation during development was associated with downregulation of parathyroid hormone 2 (pth2), a peptide associated with social isolation and mechanosensation in other aquatic larvae. This work contributes to a growing body of literature implicating miRNAs in developmental neuroplasticity, presenting precedented and novel signatures of the transcriptional and regulatory response to isolation.

Many animals navigate their world largely by seeing and feeling it. To disentangle these visual and mechanosensory contributions, we developed a virtual reality assay targeting the optomotor response in adult wild-type zebrafish swimming against flow. By projecting dynamic visual patterns onto the walls of a variable-speed flow tank, we decoupled wide-field optic flow from hydrodynamic velocity. We then tested fish responses to abrupt visual perturbations while they held station in the unsteady wake behind a bluff body. These perturbations reliably elicited compensatory optomotor responses, with fish aligning to the direction of the moving stimulus. Notably, this behavior was absent in uniform flows, suggesting that fish prioritize visual input when predictive lateral line signaling is compromised. We propose that this sensory shift serves to optimize swimming energetics in turbulent wakes. Extending this framework, we further show that zebrafish swimming against flow, whether alone or in groups, exhibit heightened escape responses to looming visual stimuli. Together, our findings reveal that fish sensory strategies are not fixed but dynamically tuned to hydrodynamic context: favoring visual cues in turbulent environments and lateral line input in uniform flows. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/715425v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d7ba00org.highwire.dtl.DTLVardef@1f456f1org.highwire.dtl.DTLVardef@7826c4org.highwire.dtl.DTLVardef@391a68_HPS_FORMAT_FIGEXP M_FIG C_FIG