Integrative and Comparative Biology

Extensible body structures have evolved repeatedly across animals, yet the mechanisms underlying the deployment of extensible organs often remain unknown. Eristalinae hoverfly larvae (rat tailed maggots) possess exceptionally elongated posterior respiratory siphons, but the mechanism underlying their extension has not been experimentally investigated. Here, using wild collected Helophilus virgatus larvae, we show that posterior siphon extension is achieved through a folding unfolding mechanism revealed by fluorescence labeling. Phalloidin staining further demonstrated that, unlike Episyrphus sp. and Drosophila melanogaster, H. virgatus possesses a dense array of transversely oriented muscle fibers in the posterior siphon. Behavioral analyses further revealed that the posterior siphon functions not only in respiration but also as a propulsive organ for near surface locomotion through asymmetric rowing. Together, our findings identify the structural and kinematic basis of posterior siphon deployment and demonstrate how a specialized respiratory organ can evolve into a multifunctional appendage that supports both respiration and locomotion.

The mass-rearing of black soldier fly (Hermetia illucens) larvae (BSFL) is a promising solution for converting organic waste into high-quality insect protein, but preventing larval escape from open-top rearing containers remains a major management challenge. Conventional escape-control methods are often unreliable or impractical. To address this, we developed and evaluated a novel physical barrier, the anti-climbing tape, featuring regularly arranged macroscale protrusions designed to disrupt larval locomotion on vertical surfaces. We conducted a series of experiments to examine the design parameters of the anti-climbing tapes, including the gap size between protrusions and the number of protrusion rows. Our results demonstrate that the anti-climbing tape prevents escape via a dual mechanism: (1) physical obstruction, in which gaps narrower than the larval body width block larvae from passing through, and (2) adhesion reduction, in which the elevated protrusion array decreases the effective contact area for wet adhesion while increasing gravitational torque acting on the larval body. The effectiveness of these mechanisms was dependent on larval size. A design featuring 0.5-mm gaps and a 15-row protrusion array completely prevented the escape of later-instar larvae (>10 mm) in a 20-day large-scale trial, whereas the method was less effective for smaller larvae. In conclusion, the anti-climbing tape provides a robust and chemical-free approach to BSFL escape in mass rearing. To ensure reliable performance, its design parameters, both gap size and array width must be optimised to suppress the mechanical and adhesive components of larval climbing according to the target larval size. Conflict of interestS. Jang and M. Shimoda are inventors on a Japanese patent application (No. 2022-172252, filed November 27, 2022) related to the method described in this manuscript. FundingThis study was supported by Korea-Japan Joint Government Scholarship Program for the Students in Science and Engineering Departments, the Korean Scholarship Foundation, and the University of Tokyo Foundations Support Fund for International Students.

Laws of physics shape morphological and behavioral adaptations to locomotion at different body sizes. Water striders serve as a model taxon to study how simple physical constraints of water-surface habitats affect their behavior and morphology, and hydrodynamics of rowing by midlegs on the surface is well understood. However, the physics of the subsequent passive sliding has been less explored. We created a model of sliding on the water surface to simulate the effect of body mass, striding type, and wetted leg lengths on an insects ability to float on the surface and on the sliding resistance. The model predicts that to support their weight on the surface during sliding, the heavy species should either develop long forelegs that support the frontal part of its body during symmetrical striding (when two midlegs thrust) or use asymmetrical striding (when one forward-extended midleg supports the body while the other midleg and contra-lateral hindleg thrust). These predictions are confirmed by the behavior and morphology of various Gerridae species. Hence, the results illustrate how simple physical processes specific to a certain habitat type have far-reaching consequences for the evolution of morphological and behavioral diversification associated with body size among biological organisms in these habitats.

How animals jump and land on a variety of surfaces is an ecologically important problem relevant to bioinspired robotics. We investigated this topic in the context of the jumping biomechanics of the planthopper Lycorma delicatula (the spotted lanternfly, SLF), an invasive insect in the US that jumps frequently for dispersal, locomotion, and predator evasion. High-speed video was used to analyze jumping by SLF nymphs from take-off to impact on compliant surfaces. These insects used rapid hindleg extensions to achieve high take-off speeds (2.7-3.4 m/s) and accelerations (800-1000 ms-2), with midair trajectories consistent with zero-drag ballistic motion without steering. Despite rotating rapidly (5-45 Hz) in the air about time-varying axes of rotation, they landed successfully in 58.9% of trials; they also attained the most successful impact orientation significantly more often than predicted by chance, consistent with their using attitude control. Notably, these insects were able to land successfully when impacting surfaces at all angles, pointing to the emerging importance of collisional recovery behaviors. To further understand their rotational dynamics, we created realistic 3D rendered models of SLFs and used them to compute their mechanical properties during jumping. Computer simulations based on these models and drag torques estimated from fits to tracked data successfully predicted several features of their measured rotational kinematics. This analysis showed that SLF nymphs are able to use posture changes and drag torques to control their angular velocity, and hence their orientation, thereby facilitating predominately successful landings when jumping. SummaryHigh-speed video revealed that juvenile spotted lanternflies are adept at landing after tumbling rapidly midair during jumping. We present computer simulations and realistic 3D models to help explain these abilities.

Insects exhibit remarkable adaptability in their locomotive strategies across diverse environments, a crucial trait for foraging, survival, and predator avoidance. Microvelia, tiny 2-3 mm insects that adeptly walk on water surfaces, exemplify this adaptability by using the alternating tripod gait in both aquatic and terrestrial terrains. These insects commonly inhabit low-flow ponds and streams cluttered with natural debris like leaves, twigs, and duckweed. Using high-speed imaging and pose-estimation software, we analyze Microvelia spp.s movement across water, sandpaper (simulating land), and varying duckweed densities (10%, 25%, and 50% coverage). Our results reveal Microvelia maintain consistent joint angles and strides of their upper and hind legs across all duckweed coverages, mirroring those seen on sandpaper. Microvelia adjust the stride length of their middle legs based on the amount of duckweed present, decreasing with increased duckweed coverage and at 50% duckweed coverage, their middle legs strides closely mimic their strides on sandpaper. Notably, Microvelia achieve speeds up to 56 body lengths per second on water, nearly double those observed on sandpaper and duckweed (both rough, frictional surfaces), highlighting their higher speeds on low friction surfaces such as the waters surface. This study highlights Microvelias ecological adaptability, setting the stage for advancements in amphibious robotics that emulate their unique tripod gait for navigating complex terrains.

Dispersal over geographic barriers plays an essential role in colonization, gene flow, metapopulation dynamics, and invasion (Bowler and Benton 2005). Since dry lands strictly separate freshwater habitats, as expressed by the phrase "islands of water in a sea of dry land" (Faulks, Gilligan, and Beheregaray 2010), dispersal among freshwater waterbodies by themselves is challenging of aquatic organisms. Rumors have existed worldwide that freshwater fish eggs are dispersed by attaching to (ectozoochory) or excretion from (endzoochory) waterbirds (Hirsch et al. 2018). It is well documented that waterbirds disperse aquatic plants, zooplankton, and various aquatic invertebrates, which are co-distributed with fishes (Green et al. 2023). However, there is only two reported cases of empirical evidence of endzoochory in freshwater fishes and no scientific evidence of ectozoochory (Hirsch et al. 2018; Silva et al. 2019; Lovas-Kiss et al. 2020; Green et al. 2023). Here, we show that the southern medaka (Oryzias latipes, hereafter medaka) egg can travel passively by attaching to waterbirds.

Because Strepsiptera can fly vertically from a standing start, at least [1/4] of their body mass must be dedicated to flight muscle. Adult male Strepsiptera also do not feed and die within a few hours of eclosing, so much normal adult insect anatomy has been discarded, leading to a flight muscle to total mass ratio (FMR) of at least 30%--this is medial for Hymenoptera, but as the lower bound for Strepsiptera, it indicates substantial aerial ability. On account of their high FMR and low wing loading, Strepsiptera are capable of widely varied flight. Moreover, the often incongruous descriptions thereof (that they fly slowly, fly quickly, are clumsy, are graceful, etc.) are paralleled in well-established phases of sex pheromone tracking in moths. For nearly all of their brief eclosed adult lives, male Strepsiptera are airborne, for which they are well-adapted. Correspondingly, strepsipteran propagation is utterly dependent on flight. Thus, flight is the lens through which much strepsipteran ecology is clarified. Accordingly, I photographed free-flying Triozocera texana (nocturnal) in the field and analyzed the images. Strepsipteran wings are remarkably flaccid and potentially teneral, leading to certain flight advantages. At night, spatial acuity is especially poor in tiny insects, but halteres apparently compensate so well that even later derived diurnal Strepsiptera identify calling females chemotactilely--not visually--and shun resolution for high sensitivity. Future directions are discussed, as well as experimental techniques that are problematic when applied to Strepsiptera.

Male crickets engage in intense aggressive behavior, competing for resources. In this study, we focus on the quick movements during tactile combat in the cricket fight, to understand how they defeat the opponents. We performed kinematic analysis following high-speed cam recording of the fight. High-speed cam recordings showed that the attacker jumped to the head of the attacked cricket and thrusted it backwards. The attacked cricket was sometimes flipped over and tended to retreat. To understand how the attacker jumps effectively to flip over the opponent, we compared the attack-jump and escape-jump. The kinematics analysis demonstrated that the attack motion is different from the jump in the case of escaping from threats. The attacker cricket adjusted the direction of its body using its forelegs. The mandibles were used to hook onto the head of the attacked cricket. The attacked cricket moved its hindlegs with different kinematics to jump in the case of escape and exerted greater velocity. These findings advance our knowledge of how animals utilize their body depending on the situation.

Reliably capturing transient animal behavior in the field and laboratory remains a logistical and financial challenge, especially for small ectotherms. Here, we present home camera systems as affordable, accessible, and suitable alternatives for monitoring small, cold-blooded animals historically overlooked by commercial camera traps. Home security cameras are often weather-resistant, operate offline or online, and allow collection of time-sensitive behavioral data in laboratory and field conditions with continuous data storage for up to four weeks. These lightweight cameras can also utilize phone notifications over Wi-Fi, alerting users when animals enter a space of interest and enabling sample collection at proper time periods. We present our findings, both technological and scientific, in an effort to elevate tools that enable researchers to maximize use of their research budgets. We discuss the relative affordability of our system for researchers in South America, home to the largest ectotherm diversity.

Centipedes coordinate body and limb flexion to generate propulsion. On flat solid surfaces, the limb-stepping patterns can be characterized according to the direction in which limbaggregates propagate, opposite to (retrograde) or with the direction of motion (direct). It is unknown how limb and body dynamics are modified in terrain with terradynamic complexity more representative of their natural heterogeneous environments. Here, we investigated how centipedes that use retrograde and direct limp-stepping patterns, S. polymorpha and S. sexspinosus, respectively, coordinate their body and limbs to navigate laboratory environments which present footstep challenges and terrain rugosity. We recorded the kinematics and measured the locomotive performance of these animals traversing two rough terrains with randomly distributed step heights and compared the kinematics to those on a flat frictional surface. S. polymorpha exhibited similar body and limb dynamics across all terrains and a decrease in speed with increased terrain roughness. Unexpectedly, when placed in a rough terrain, S. sexspinosus changed the limb-stepping pattern from direct to retrograde. Further, for both species, traversal of rough terrains was facilitated by hypothesized passive mechanics: upon horizontal collision of a limb with a block, the limb passively bent and later continued the stepping pattern. While centipedes have many degrees of freedom. our results suggest these animals negotiate limb-substrate interactions and navigate complex terrains, by offloading complex control and leveraging the innate flexibility of their limbs.

Two Vorticella species undergo a synchronous transition from sessile zooids to motile telotrochs, which swarm enveloped in secreted mucus, subsequently forming dense aggregations on substrates and reverting to the zooid form. This cyclical process recurs on a daily basis. Each species exhibits a unique mode of swarming behavior. We hypothesize that these behaviors may serve to facilitate efficient feeding while concurrently acting as a mechanism for predator avoidance.

During their period of summer dormancy, Australian Bogong moths Agrotis infusa undertake seemingly random evening flights, filling the air with densities in the dozens per cubic metre. The purpose of these flights is unknown, but they may serve an important role in Bogong moth navigation, which remarkably enables them to return to the same exact summer sites-- generation after generation--after migrating around 1000 km, and with no opportunity to learn their route or destination from prior generations. The recent development of the camera-based insect monitoring method, Camfi, enables quantitative observations of Bogong moth behaviour at an unprecedented scale. To gain a better understanding of the summer evening flights of Bogong moths, we have extended Camfi to facilitate automated video tracking of flying insects, taking the already-high throughput of the method to a new level. We used this new method to record the evening flight behaviour of Bogong moths in two elevational transects below the summit of Mt. Kosciuszko, NSW, on a single night in February 2021, and found that these flights were not random, but were systematically oriented in directions relative to the azimuth of the summit of the mountain. These results stimulate interesting and plausible hypotheses relating to previously unexplained summer evening flight behaviour of Bogong moths, and the mechanisms of their long-distance navigation.

Animals continuously evaluate environmental cues to guide approach-avoidance decisions, with internal states like hunger dynamically shaping how stimuli are acted upon. While most studies examine the valence-switching of stimuli from appetitive to aversive using simplified or ambiguous stimuli, we leveraged a system in which a single prey contains both appetitive and aversive features. The nudibranch Berghia stephanieae, is a specialist predator of the sea anemone, Exaiptasia diaphana. These nudibranchs must resolve conflicting signals where chemical cues signal food, while contact can result in injury or death. The danger posed by Exaiptasia was described and quantified through direct counts of nematocysts fired into Berghia and multiple instances where the Berghia was captured and consumed by its prey. To test how internal state influenced the perception of stimuli from prey we recorded predatory behavior of Berghia after different periods of food deprivation. We found that the olfactory cues from prey were attractive to Berghia, even when animals were sated, and usually led to a contact-mediated investigation of prey. Hunger independently modulated olfactory and contact cue valence at different internal states and time scales of food deprivation. Hunger specifically altered the threshold for avoidance following contact with prey, indicating that somatosensory and chemotactile cues are modulated by hunger unlike olfactory cues. Our results highlight how internal state and sensory modality interact to shape decision making in a biologically relevant, high-risk predation context.

Despite its ubiquity in natural flows, the effects of turbulence on fish locomotion and behavior remain poorly understood. The prevailing hypothesis is that these effects depend on the spatial and temporal scales of the turbulence relative to the fishs size and swimming speed. But in conventional facilities, turbulence usually increases with mean flow, which forces higher swimming speeds and can leave these relative scales unchanged. We therefore present a novel experimental facility that leverages a jet array to decouple the turbulence from the mean flow and systematically control its scales. This approach allows the ratio of turbulent to fish inertial scales to be varied over an order of magnitude, providing a controlled framework for quantifying fish-turbulence interactions. The facility also supports experiments probing strategies fish may use to cope with turbulence, including collective behaviors. Insights from this work have broader implications for ecological studies and engineering applications, including the design of effective fishways and bio-inspired underwater vehicles.

Odor-plume-guided navigation, tracking an odor plume to its source, is a primordial behavior used by most animals to search beyond the visual range. Here we report the first laboratory observations of octopuses performing this behavior, demonstrating that they can use odor plumes to find food. In a three-station discrimination task carried out in the dark, octopus showed a strong preference to move upstream towards the food-baited target, supporting the hypothesis that they are performing odor-guided search. When seeking a single baited target, also in the dark, octopuses not only preferred to move upstream towards the food source, but they also displayed characteristic motions associated with odor-gated rheotaxis, a commonly used odor tracking strategy used by many animals, which includes pausing, switchbacks, and across-stream redirections to the bait. Additionally, when approaching single baited stations the octopus often made reactive fast lunging motions. The observation of these fast arm-aligned motions (FAAM), taken together with the observation that the octopus did not have a characteristic body axis orientation to the bait, as would be expected if bilaterally symmetric organs such as the olfactory pits guided this behavior, supports the hypothesis that the suckers are the primary chemosensory organs driving octopus odor-guided behaviors.

The control and stability of flying and swimming animals is typically determined by measuring their responses to discrete gust perturbations. For the rigorous measurement and analysis of such responses, it is necessary to generate gusts that are precise, controllable and repeatable. Here, we present a method to generate discrete gusts under laboratory conditions using a vortex ring. Unlike other methods of gust generation, the vortex ring can be well characterized and is highly controllable. We first outline the theoretical basis for the design of a gust generator, and then describe an apparatus that we developed to generate discrete gusts. As a case study, we tested the efficacy of this method on freely-flying soldier flies Hermetia illucens. The method described here can be used to study diverse phenomena ranging from natural flight and swimming in insects, birds, bats and fishes, to the artificial flight of drones and micro-aerial vehicles.

Systematic descriptions of the underlying interaction rules that insects use to support group and swarm flight has the potential to contribute to mathematics, biology, and robotics, including aerial swarming under sensory and computational limitations. This study analyzes 1,000 trajectories of flying honeybees in crowded conditions approaching a moving stimulus and finds how during this stimulus, honeybees coordinate flight through pairwise interactions involving a novel three-zone decision-making process. The experimental setup consists of 3-D position reconstructions via a high speed camera system recording honeybee foragers returning to a hive entrance actuated to move robotically. The analysis consists of neighborhood identification through three methods (cross-correlation, distance threshold, and average distance threshold), which reveals the dominant interaction is pairwise. The individual leader-follower pair interactions are then tested against three regulation candidates: optic flow, relative velocity, and optical expansion rate, based on minimizing root mean square error. The results show that each follower demonstrates a three stage process involving a feedback rule change, linked by an intermediate observation/decision phase. During the initial "lock" phase, an insect maintains a consistent optical expansion rate until inter-agent distance closes to 10 cm. The regulation candidates then undergo large variations during a relatively long observation/decision zone, with 1.04 seconds being the average time in the decision zone. 79% of the paired insect entries into the decision zone result in subsequent re-engagement to track the same initial leader, while 21% result in disengagement from the group behavior. Visual regulation candidate comparison in the third stage indicates that upon re-engagement, the follower relative velocity is regulated to provide consistent velocity matching between agents. The third stages velocity tracking is consistent with a closed-loop feedback proportional-integral (PI) controller regulating velocity tracking error. Across the insect population studied, the proportional gain remained showed minimal variability over individuals, a derivative gain was considered and found negligible, and the integral gain varied by individual. Collectively, these findings underscore the existence of an alternative swarm architecture, highlighting individual decision-making capabilities, feedback regulation target changes, and the presence of reactive, deliberative, and moderate (PI control) timescale interaction rules contained within aerial groups.

Despite much of the literature perceiving fish schooling as an organized system with a focus on fixed formations for theoretical analyses, experimental observations suggest that frequent positional rearrangement commonly occurs. Previous studies have also demonstrated that fish schools reduce locomotor costs relative to individuals swimming alone. This introduces an intriguing dichotomy. How can individual fish within schools exhibit dynamic interactions while also saving energy? We hypothesize that schooling dynamics are the result of positional and kinematic modulation of individuals responding to fluid dynamic stimuli from the movement of neighbouring individuals. We propose a two-tier approach to studying kinematic modulation within fish schools. First, quantification of the variation of individual movement in a school relative to that of a solitary individual uses an analytical pipeline combining artificial-intelligence-enabled tracking and video processing. Second, the study of kinematic modulation in response to hydrodynamic stimuli uses a mechanical flapping mechanism coupled with an enclosure to control fish position. We discovered that fish in schools exhibit higher levels of positional and kinematic modulation than individuals swimming alone. Fish swimming in enclosures can robustly respond to fluid stimuli from either a simple robotic fish or other fish located in proximity. This two-tier approach allows high-resolution analysis of positional and kinematic modulation within fish schools and their impacts on energy conservation resulting from collective movement.

The evolution of early land vertebrates from aquatic forms of life was a biological milestone. The transition to land was accompanied with expectedly challenging physiological and morphological evolutionary hurdles. So far, fossil records have provided substantial information on the origin of quadrupedal locomotion. However, fossil evidence alone is insufficient to understand how the soft-tissue-dependent motor functions and locomotion were acquired and developed. In the present study, we focus on locomotion of the sturgeon, an extant primitive fish, as a new experimental model, to investigate behavioural plasticity. Their locomotion in low-water-level conditions was similar to an escape response in water, the C-start escape response, which is used by most fish and amphibian juveniles to avoid predation. Sturgeons were also found to have mastered rolling-over in response to low water levels, resulting in the improvement of their trunk-twisting action. Sturgeons acquired an efficient shift in their centroid, thereby improving their mobility. We hypothesise that the escape response triggered by environmental hazards drove the development of locomotion, which was accompanied by a variety of behaviours.

Predator-prey interactions shape the evolution of escape behavior in prey, including different combinations of evasive movements, that may enhance unpredictability in fleeing directions and trajectories. So-called protean motion can enhance survival of flying prey in the wild, but quantifying such behaviors under natural conditions remains challenging. Here we used stereoscopic high-speed videography to record the escape flight behavior of wild males of the butterfly species Morpho menelaus in the Amazonian rainforest, and reconstructed 3D flight trajectories using artificial-neural-network-based tracking. During the experiments, we used a lure to attract freely patrolling male butterflies and elicited escape flights by intercepting their trajectory with a looming insect net swing. We then compared the escape flight kinematics to the pre-attack patrolling behavior. Attacks first induced a rapid upward maneuvering, directly followed by an unpredictable horizontal turn. The following escape flight trajectories showed increased horizontal erraticity and greater intra-individual heading variability, as compared to the pre-attack flight. Surprisingly, the mean speed decreased in the escape phase, notably in the horizontal plane. A significant negative association between horizontal trajectory complexity and flight speed was detected, indicating a speed-erraticity trade-off. These results show that wild Morpho butterflies respond to attacks by combining a climbing maneuver with an unpredictable heading change, followed by a protean escape flight; this increased escape erraticity comes at the expense of reduced escape flight speed. Because these large and relatively slow-flying butterflies display bright iridescent blue coloration on their dorsal wing side, erraticity during flight might enhance the dynamic flash coloration, likely limiting accurate targeting by predators.