Integrative and Comparative Biology

Many arthropods (insects and crustaceans) rely on their antennular chemosensory system to detect key environmental resources like food. While odor mediated food search is well studied in insects, characterization of crustacean chemosensory behavior has been limited by the long lifespans and large size of traditional crustacean model species. Here, we report the first characterizations of the food search behaviors of the genetically tractable amphipod crustacean, Parhyale hawaiensis. We find that Parhyale can locate an odorous food pellet, and predominantly approach food using direct, targeted swims from the arena walls. Removal of both first and second antennae dramatically reduced foraging success and impaired Parhyales ability to control take-off angle and maintain a stable heading during swims. Removal of the first or second antenna alone did not significantly disrupt foraging, and resulted in mild disruption of orientation phenotypes. Intact animals performed sharp turns near the location of the food pellet, which were observed when either first or second antenna were present, but not when all antennae were removed. Turns were longer and had higher average angular velocities following removal of either set of antennae, with full antenna removals representing the most extreme phenotype. In contrast with the long-held theory that the crustacean second antennae exclusively mediate contact chemosensation, we report that first- and second- antennae both contribute similarly to food localization and stabilization of locomotion in Parhyale in our behavioral paradigm. This work establishes Parhyale as an accessible model for studying olfactory behaviors in an aquatic arthropod.

Argentine ants (Linepithema humile) utilize adhesive pads (arolia) to climb smooth surfaces. Previous research found that ants can adjust their individual arolium engagement according to their locomotion mode. However, it remains unclear how they distribute arolium engagement across multiple limbs to climb effectively, and how arolium engagement varies within a climbing step. As the arolium is a well-known adhesive organ, we hypothesized that engagement across different legs is distributed according to the normal forces required for balancing the body during climbing. To test this, we measured Argentine ants' arolium engagement on a vertical glass surface using a Frustrated Total Internal Reflection (FTIR) sensor and compared it to the required normal forces from a quasi-static model. Contrary to the required normal force, the measured arolium engagement was asymmetric between upward and downward climbing, and changed over time. Our results indicated that the quasi-static force requirements are not sufficient to explain arolium engagement in climbing Argentine ants, and suggested that other factors, such as body dynamics, ants' anatomy and behavioral preferences, should be included.

The ability of animals to adjust their heading, i.e. to turn, is essential for all walking animals. While several studies have addressed how leg movement or inter-leg coordination may change during turning, relatively little is known about how turning-related changes scale with turn magnitude. Here, we used spontaneous and visually induced turns of unrestrained walking stick insects to test (i) how high-level parameters of unrestrained turning scale with low-level parameters of leg movement, and (ii) the effect of visual guidance on turning parameters. To this end, we used a step change in stationary landmark position in an open-field arena to constrain timing and magnitude of target-directed turns. These visually guided turns were compared with spontaneous turns in an all-white condition. We show that visually induced turns were walked at a larger forward velocity and had fewer short steps than spontaneous turns. The scaling of turning responses was dominated by an increase in turning duration (factor 1.87) rather than turning speed (factor 1.32). Increased rotational velocity correlated with reduced forward velocity, though with flexible timing of both effects. These changes were accompanied by larger shifts in step direction, as well as an increased asymmetry of step types between inner and outer legs, suggesting a mix of distinct turning strategies, that depend on overall turn angle. Future models on six-legged locomotion should thus consider the incorporation of more than one mechanism to govern turning.

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

Dung beetles can use a variety of orientation cues to maintain a consistent bearing during ball-rolling. Where several cues are available, they appear to learn the spatial relationship between them, providing redundancy if some cues are removed. Mounting evidence indicates that such a learning process is implemented in the insect head direction circuit; specifically, in the plastic substrate between sensory input neurons and compass neurons in the central complex. This plasticity appears to be driven by rotational movements, providing a clear link with observed beetle 'dance' behaviour. Here, we extend our functional model of this circuit and use it on a robot platform, to test it in the same behavioural assay as was used for the beetles. The robot was able to replicate the beetle's ability to substitute a directional wind cue for a point source light cue in guiding straight-line movement. However, it also revealed significant biasing coupled to dance direction. This biasing appears to be caused by inherent conflict between recurrent and instantaneous inputs to the compass circuit. We predict that the real insect should experience similar issues unless it has evolved a neural mechanism to compensate.

Artificial light at night (ALAN) is a widespread anthropogenic phenomenon with varied physiological, behavioural, and ecosystem-level effects. Its impacts have been studied extensively at the population level, however less is known about the individual changes that underpin these larger trends. We use a networked video system combined with GryllAI--a deep learning-based system for continuous individual monitoring--to explore this in the field cricket, Gryllus campestris. Applying field-realistic artificial light (10-25lx) or a control treatment to burrows, we continuously track the activity of 144 nymphs across >38,000h of video footage, recording life history outcomes for each individual. Results indicate that ALAN exposure does not influence daily activity timing, total activity duration, predation risk, or nymphal development duration. However, changes in fine-scale behaviour were apparent, with ALAN causing crickets to enter and exit burrows less frequently, especially at night; and spent greater proportion of time outside burrows during daytime. These behavioural adjustments were not evident from broad scale activity trends that could be observed manually. Consequently, our findings suggest that aggregate measures of activity may fail to capture the full scope of ALAN-mediated impacts in nature, and that automated monitoring techniques offer a promising means of addressing this.

Collective movement in social organisms emerges from local interactions and can generate large-scale spatial patterns of ecological relevance. In termites, trail formation is a well-known collective phenomenon, yet reproducing and recording its emergence under controlled laboratory conditions using whole colonies remains challenging. Existing laboratory approaches often rely on confined arenas or manually assembled subgroups, which can restrict movement and limit observation of colony-level dynamics. Here, we present a semi-folded arena designed for whole-colony observation of termite movement under controlled conditions. We developed a circular semi-folded arena that remained continuously connected to an intact nest and allowed individuals to move across a central observation surface while recirculating through a folded peripheral section. Using whole colonies of the Neotropical termite Constrictotermes cyphergaster, we recorded exploratory activity under baseline conditions, in the absence of added food or water. High-resolution video recordings were analysed using automated movement extraction to recover trajectories and visualise collective trail structure. Within the first 6 min of activity, collective trail structure was observed in 15 of the 16 colonies analysed. Under these conditions, the semi-folded setup captured early collective trail structure, visible as convergence of cumulative trajectories along shared routes radiating from the arena entrance region. Automated movement extraction was compatible with dense whole-colony recordings and yielded large quantities of positional data during the initial observation interval. Descriptive trajectory-based outputs, including speed distributions for workers and soldiers, showed that the recordings were suitable for recovery of fine-scale movement information. Repeatedly used routes were also often marked by visible dark traces on the paper lining by the end of the observations, providing a qualitative record of cumulative route use. The semi-folded arena provides a practical method for recording whole-colony termite movement under laboratory conditions while maintaining continuous nest access and avoiding manual transfer of individuals during trials. Rather than replacing conventional arena designs, this approach offers an additional methodological option for studying emergent movement patterns in species for which whole-colony observation is feasible. More broadly, it expands the experimental toolkit available for investigating colony-scale spatial organisation under controlled conditions.

Colour and contrast are commonly deployed in anti-predator signals like aposematism or deimatism. In oceans, colour information diminishes with depth, leaving blue bioluminescence the most common visual signal, regardless of function. Bioluminescence can deter predators, but without contrasting colours, how so is largely uncharacterized. Here, we test this by observing fish predators responding to prey that use defensive bioluminescence (Ostracods, Cypridinidae). By manipulating potential chemical defences of prey, and by comparing feeding responses to both luminescent and nonluminescent prey, we show that luminescent prey are unpalatable and use facultative bioluminescence as an aposematic signal. We observed active, luminescent prey secrete bioluminescence only after being attacked. Predatory fishes rarely consumed luminescent prey, especially compared to nonluminescent alternatives. Food treatments revealed that luminescent species may possess some unidentified defence over nonluminescent relatives because fishes also readily ate luminescent prey that had been treated (frozen or boiled), which removed such defences. Over the course of four experimental trials, predators were less likely to consume luminescent prey as their cumulative exposure to anti-predator light displays increased, indicative of learning. Despite their intermittency, temporally dynamic signals like aposematic bioluminescence may be as common and effective as better-studied static coloration, especially in marine ecosystems.

Nutritional symbionts can be essential for their animal hosts. The bacterial symbiont of the leaffooted bug, Leptoglossus zonatus, Caballeronia, is acquired from the environment each generation in the 2nd instar. The symbiont is critical for L. zonatus: aposymbiotic bugs are unable to reproduce. We hypothesized that symbiotic bugs excrete Caballeronia where juveniles might find and consume them. We inoculated L. zonatus with GFP-labelled Caballeronia and examined feces of each life stage. We found that Caballeronia is excreted almost exclusively in the adult stage. We then asked if 2nd instar nymphs could acquire Caballeronia from feces. Nymphs were provided with a) feces from adults fed GFP-labelled Caballeronia, b) GFP-Caballeronia in culture, or c) water only. We found that feces-fed bugs had similar rates of symbiont acquisition to those fed Caballeronia in culture, indicating that feces can be a source of Caballeronia for L. zonatus. However, compared to culture fed individuals, bugs fed feces had reduced survivorship and required longer to develop, and surviving adults had reduced mass. Bacterial motility assays showed that in contrast to cultured Caballeronia cells, Caballeronia in feces were non-motile. These results show suggest that feces can be a source of Caballeronia, at least in some environments, however transmission mode can influence success of the offspring.

Animals rely on a wide range of environmental signals, including celestial and terrestrial cues for navigation. While celestial cues, such as the sun, play a major role in maintaining a constant heading during long-distance migration and dispersal, terrestrial cues provide an animal with a short-range navigation system, ideal to pinpoint highly specific locations. In Monarch butterflies, the simulation of a terrestrial landmark, i.e. a vertical stripe, induces an attraction behavior (all animals head toward the stimulus) while a small green light spot, simulating the sun, elicits menotactic orientation (animals adopt individual-specific headings relative to the stimulus). However, the mechanisms underlying how the animal distinguishes between a stimulus as a terrestrial landmark versus a celestial cue remains unclear. To explore this, we tested non-migratory Monarch butterflies (Danaus plexippus) in a flight simulator. The inner surface of simulator was equipped with an area of LEDs, allowing to present different visual stimuli to the butterflies during tethered flight. By systematically manipulating the stimulus width, height, brightness, and elevation we found that Monarch butterflies exhibited attraction behavior to high contrast areas, like stripe edges. Menotactic behavior was not achieved by solely decreasing the stimulus to a small light spot but also required for the stimulus to be presented at higher elevation to be interpreted as a sun stimulus. These findings suggest that multiple parameters, inherently set by the butterflys navigation system, are critical to interpret a visual stimulus as celestial cue or terrestrial landmark, producing dynamic switches between different orientation strategies during navigation.

The queen bee is the central individual responsible for colony establishment, growth, and survival. Reliable confirmation of successful mating, continued queen presence, and normal reproductive performance is essential for effective colony management. We present a queen bee detection system based on an array of Hall-effect sensors and a miniature magnetic tag attached to the queen. The system is designed for continuous operation and real-time monitoring. A prototype was developed, constructed, and evaluated under both laboratory and field conditions. Field experiments conducted in an apiary demonstrated that the system can reliably detect queen bee passages through the hive entrance, enabling the identification of activities associated with mating flights. The results confirm the feasibility of Hall-effect sensing for automated, non-invasive queen bee monitoring and establish magnetic sensing as a promising new measurement modality for precision apiculture.

Predators are often considered regulators of disease in prey populations, a concept central to the "healthy herd hypothesis". This hypothesis suggests that by preferentially removing infected individuals, predators can reduce parasite prevalence. However, predators may also act as disease vectors, facilitating the spread of parasites. We investigated whether stickleback fish (Gasterosteus aculeatus) can act as vectors for the transmission of the obligate bacterial parasite Pasteuria ramosa to its Daphnia host, a widespread freshwater zooplanktor. We fed infected D. magna to sticklebacks, and subsequently analysed faecal samples for the presence, viability, and infectivity of parasite transmission stages (= spores). We recovered approximately 60% of the consumed spores from fish faeces and these spores did not suffer from reduced infectivity to D. magna. Additionally, spores associated with sloppy feeding did not reduce infection rates. Thus, consumption of infected hosts by fish does not eliminate the parasite, but in contrary, may contribute to the spread and persistence of P. ramosa in natural populations, potentially influencing parasite dynamics in natural freshwater ecosystems.

The accumulation of microplastics (MP; plastic particles 1 m - 1 mm in diameter) in the environment is an increasing global concern. Although the physiological effects of MP on organisms, including humans, are increasingly documented, their impacts on behaviour are far less understood. Further, little is known about how MP affect adaptive phenotypic plasticity - the ability of a genotype to adaptively modify its phenotype in response to environmental cues. Anuran tadpoles are key models for studying phenotypic plasticity, with well-established evidence for predator-induced behavioural adjustments. Tadpoles typically reduce their activity and risk-taking when exposed to chemical cues released by predators, which has been proven to be adaptive. We investigated whether MP exposure from the fertilised egg stage alters the behaviour of agile frog (Rana dalmatina) tadpoles, and whether it interferes with their predator-induced behavioural plasticity. Tadpoles exposed to chemical cues from dragonfly larvae showed the expected antipredator response: these larvae showed significantly reduced movement activity and risk-taking. Although exposure to MP did not influence the behaviour of tadpoles that had not been exposed to predator cues, and also did not alter predator-induced changes in movement activity, it entirely abolished the predator-induced reduction in risk-taking. These results indicate that MP can compromise antipredator behaviour, increasing the vulnerability of individuals and natural populations to predation without causing visible developmental abnormalities. We recommend that future MP research targets behavioural traits with direct relevance to survival and reproduction, and examines how adaptive phenotypic plasticity is affected.

The plasmodium of Myxogastoria (a group of amoeboid protists) species often crawls around the forest floor to feed while searching for places to form fruiting bodies for reproduction (sporulation). Certain environmental factors that trigger sporulation have been reported; however, other unknown factors are also expected. In this study, we reported field observations of Physarum rigidum and Fuligo septica. Inspired by the field observation, we examined the effects of multiple factors on sporulation in laboratory experiments using Physarum polycephalum. We found that:(1) there was a critical body size below which sporulation did not occur under our experimental conditions and (2) the plasmodium selected its sporulation sites from the available landscape of the experimental arena: dry and low sites for the majority and dry and high sites for the minority. Further analysis revealed that they preferred the edge area at the high site. We discuss the possible ecological importance of the threshold and location preference

Buzz pollination is an important behavior in which bees use vibrations to extract pollen from poricidal anthers. However, the extent to which vibration frequency influences pollen release remains unclear. Here, we quantified pollen expulsion from Solanum sisymbriifolium anthers subjected to harmonic excitation over a broad frequency range encompassing the anthers first natural frequency. We excited anthers to expel pollen and measured anther kinematics and pollen release using high-speed videography. Particle tracking enabled continuous estimation of pollen release throughout each buzzing event, allowing both initial pollen flux and total pollen released to be quantified. Pollen release depended strongly on excitation frequency. Initial pollen flux, total pollen release, and anther kinematics peaked when excitation frequency approached the anthers natural frequency. Anther tip velocity amplitude exhibited the strongest correlation with total pollen release (r = 0.755) and initial pollen flux (r = 0.898). Experimental observations were compared with nonlinear and linear statistical models of pollen release. While both models captured trends in normalized pollen flux, they overpredicted total pollen release, suggesting that adhesive interactions play important roles during extended buzzing events. These findings demonstrate that anther structural dynamics influence pollen release and suggest that vibration amplification may improve the efficiency of buzz pollination.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

Globally, nocturnal lightscapes are now determined by both moonlight and light pollution, or artificial light at night (ALAN). Organisms respond both to changes in moonlight across lunar cycles and to alterations in light conditions due to artificial light. The interaction of natural and artificial light is a critical aspect to incorporate into our understanding of how crepuscular and nocturnal ecology is altered in anthropogenically-modified landscapes. In this manuscript we review the rapidly expanding body of research on ecological impacts of ALAN to (1) assess patterns of lunar data inclusion and (2) summarize documented interactions of moonlight and ALAN. Three-fourths (72%) of 379 papers reviewed did not incorporate moonlight into their statistical analyses and experimental design. Only 12% directly investigated interaction effects of moonlight and ALAN. However, 70% of these studies reported an interactive effect. Considering this stark contrast, as a precursor to our literature review, we present an overview of moonlight and the lunar cycle for biologists. The overarching trend emerging from the literature is that biological impacts of ALAN decrease with increasing moonlight, although the opposite is true in some cases. After summarizing the literature, we present general hypotheses regarding the interaction of the lunar cycle and ALAN. These hypotheses consider the different forms of ALAN encountered by organisms (i.e. skyglow and light sources) and account for the influence of cloud cover. Finally, we suggest best practices for incorporating moonlight into future research on biological impacts of ALAN.

In turbulent environments, odorants from different sources arrive at different times, potentially providing cues for odor source segregation. In several invertebrate species, short differences in odorant onset enable freely moving animals to discriminate odorant mixtures. In vertebrates, however, studies of sensitivity to odorant onset asynchrony have been conducted under highly constrained sampling conditions, such as with odor delivery tightly coupled to respiration. In this study, we investigated whether domestic dogs could detect odorant onset asynchrony in odorant mixtures under conditions that preserve key features of natural odor sampling. Dogs performed a discrimination task in which odor stimuli were presented as ongoing pulse trains that began independently of animal behavior, avoiding artificial synchronization of odor delivery with sniff cycles. Dogs were trained to discriminate between mixtures of two odorants with synchronous onsets and mixtures with asynchronous onsets. Of the dogs trained, one was able to discriminate odorant onset asynchronies as short as 633 ms. Dogs also displayed sensitivity to auditory stimulus onset asynchrony, discriminating auditory asynchronies as short as 30 ms. These results provide the first demonstration of temporal sensitivity in canine olfaction and the first evidence that vertebrates can use odorant onset asynchrony under conditions that permit free odor sampling.

For oviparous animals, the decision of where to lay eggs is critical, as offspring remain sessile from oviposition through hatching and are thus unable to escape unfavorable conditions. Consequently, females are expected to select oviposition sites that benefit embryo development and survival. This may be particularly relevant for arboreal frogs, which typically lay eggs on leaves overhanging water, where embryos are exposed to predation, desiccation, and other risks until hatching. Yet studies directly linking maternal substrate choice to embryo survival remain scarce. Here, we examine how oviposition substrate influences embryo survival in the Emerald glass frog (Espadarana prosoblepon), a species in which females deposit eggs on multiple substrates, providing a rare opportunity to test how oviposition decisions affect reproductive success. Monitoring clutches in situ, we compared microclimatic conditions, hatching success, and sources of embryo mortality between the most used substrates: the spike moss Selaginella diffusa and leaves. Additionally, we conducted a two-choice experiment in semi-captivity to test whether females preferentially select one substrate over the other. Although microclimatic conditions did not differ between substrates, hatching success was significantly higher on S. diffusa, which also experienced less predation. In the two-choice experiment, all females laid their eggs on S. diffusa, and those clutches had higher hatching success and faster embryonic development rates than those on leaves. Together, these results support the hypothesis that non-random oviposition site selection in E. prosoblepon is driven by the maximization of embryo survival, demonstrating that substrate choice has measurable fitness consequences for the offspring.

Adult mosquitoes rely on plant-derived sugars for survival, reproduction, and flight, yet the plant taxa that mosquitoes encounter in nature and the odors that make those plants attractive remain poorly understood. Most studies of mosquito attraction to plant odors have focused on candidate plants selected a priori, rather than plants linked to field-collected mosquitoes. Here, we combined plant DNA barcoding, semi-field behavioral assays, and volatile profiling to identify field-associated plant resources relevant to Aedes aegypti. Plant DNA recovered from mosquitoes collected across three Florida counties revealed broad plant associations, including 90 genera spanning 37 families, with several taxa recurring across counties or appearing prominently within particular localities. Behavioral experiments in semi-field sticky-trap assays found that five field-associated plant taxa were significantly attractive relative to blank controls, indicating that taxa associated with mosquitoes in nature can also function as attractive cues under semi-field conditions. GC-MS analyses of headspace collections from 42 plant taxa detected 211 volatile compounds and revealed substantial variation in both total emission rate and odor composition among taxa. Although several compounds, including -pinene, limonene, 4-ethylacetophenone, 2-ethyl-1-hexanol, 4-ethylbenzaldehyde, and caryophyllene, were broadly distributed across plant groups, volatile profiles differed significantly among taxa and shared compounds often occurred at markedly different proportional abundances. The five behaviorally tested taxa likewise showed both overlap and divergence, sharing 17 compounds across all five taxa while differing in dominant constituents and total emissions. Together, these results show that Ae. aegypti interacts with a diverse set of plants in the field, and suggests nectar-seeking is shaped not simply by plant identity or total odor abundance, but by the composition and proportional structure of plant odors.