Integrative And Comparative Biology

Arboreal insects have developed various strategies to navigate their discontinuous habitats. Many insects, including leafhoppers, katydids, and praying mantises, exhibit the ability to actively leap across their leafy platforms and land on a distant substrate. This behavior is especially important for non-winged insects, including nymphal forms of winged insects, which cannot fly between these substrates. To make a targeted jump, an animal must first orient towards the target, estimate the target distance and angular location, and jump with the appropriate take-off speeds and angles to land on their intended substrate. In three-dimensional space, jumping from one point to another requires estimating distance, as well as azimuthal and elevational angles. Jumping insects such as mantises typically reorient their bodies on the substrate to align with the azimuthal direction of the target. This behavior effectively reduces the task to a two-dimensional problem, in which they must estimate only the distance to the target and its elevational angle. Many insects, including praying mantises, perform rhythmic lateral head movements called peering before performing a targeted jump. Although previous studies suggest that mechanisms such as motion parallax while peering are used for distance estimation, the full repertoire of behaviors that enable mantises to jump to arbitrarily located substrates remains unclear. We hypothesized that mantises have distinct behaviors for distance and elevation angle estimation, which enable them to independently modulate their take-off speeds and angles before jumping. To test this hypothesis, we developed behavioral assays in which mantises were placed on a launch platform and jumped to a target platform positioned at variable distances and angles. Using this apparatus, we filmed the jumps of Giant Asian mantis nymphs (Hierodula spp.) with high-speed videography and tracked body parts to quantify take-off speed and angle. Because mantis jumps are ballistic, their trajectories can be modeled as projectile motion. Our results indicate that mantises estimate target distance and elevation angle using two separate behavioral strategies: distance is assessed through peering maneuvers that generate motion parallax, whereas elevation angle is determined through visual fixation of the target accompanied by specific postural adjustments. By combining these behaviors, mantises modulate the magnitude and direction of propulsive force to achieve successful jumps.

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.

Multimodal communication plays a central role in animal behavior, particularly when individuals must integrate information from different sensory channels to make rapid decisions. In aquatic environments, chemical and visual cues differ markedly in their spatial and temporal properties, such that chemical signals may be constrained by limited spatial resolution and temporal instability, potentially requiring visual information to reliably guide social decisions. In decapod crustaceans, both cue types are known to mediate reproduction, yet their relative contribution to mate-location behavior remains unclear. Here, we tested how visual and chemical cues from males influence mate-location behavior in females of the prawn Macrobrachium rosenbergii. Females were placed in a central arena and exposed to four stimulus configurations combining visual cues (a life-size photograph of a male or a control background) and chemical cues (water from an aquarium with or without a male). Attraction was quantified as the time spent in each half of the arena. Females showed no directional preference when exposed to chemical cues alone or when visual and chemical cues were spatially incongruent. In contrast, females spent significantly more time near male-associated stimuli only when visual and chemical cues were spatially congruent. These results indicate that mate-location behavior in this species depends on multimodal integration with a strong contextual dependence on visual information, which appears to gate the effectiveness of chemical cues. Spatially congruent multimodal signals are therefore necessary to guide orientation during mate search, suggesting that disruption of visual or chemical information in aquaculture systems may impair mating efficiency.

Within species, individuals of the same age can differ in size. Previously, parental genetics, nutrition, space, and social interactions have been suggested to explain different growth rates. However, direct effects of larger individuals on the physiology and growth of smaller individuals are poorly understood. In this study, we investigated how larger individuals of the marine worm Platynereis dumerilii can impact the growth of smaller conspecifics. Comparing growth distributions in communally and individually reared worms, we show that larger worms suppress the growth of smaller ones. Furthermore, we were able to demonstrate that this suppression is chemically mediated. The chemical cue does not originate from faeces but is water soluble, stable for several days and smaller than 3 kDa. Our findings highlight the importance of non-reproduction related chemical signalling, showing evidence that dominant individuals can chemically suppress the growth of their conspecifics. This study provides new insights into how hierarchy can be established and maintained in a population and is particularly relevant for the growing community studying this model species.

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Locating, tracking, and intercepting objects is a fundamental behavior for many organisms. For instance, predators must track and capture erratically moving prey for their survival. Using the echolocating bat as a model species, we investigate how short-term changes in target motion predictability affect longer-term motor plans when tracking a prey item. We used a paradigm where prey motion is under experimental control, and then applied computational methods to characterize how target motion predictability influences short- and long-term behavioral control. We find that target motion predictability during the tracking phase of insect capture influences both short-term changes in sonar call control, as well as longer-term behavioral control for transitioning between hunting phases. For changes in immediate behavioral control, bats produce more bursts of calls at a higher rate when tracking unpredictable moving prey, an indication that the bat is collecting more information about the targets motion for unpredictable than predictable trials. In terms of longer-term behavioral control, target motion unpredictability delays the transition from tracking to capture phase behaviors. We suggest that the bat does this to collect more information about target motion to time the transition from tracking to capture behaviors for hunting success. Additionally, we find the effects of target motion unpredictability are first seen as changes in the vocal motor plan and then the auditory motor plan (ear motion), hinting at a sequencing of motor changes that warrant further investigation. SummaryWhen presented with a more challenging hunting task, bats will increase their production of bursts of calls at a higher rate and delay their transition into capture behaviors.

Guinea worm disease eradication efforts may benefit from environmental surveillance methods capable of detecting infected copepod intermediate hosts in aquatic habitats. We developed a three-dimensional, spatially explicit agent-based model to examine how ecological processes influence detection probability for a hypothetical water sampling method. The results show that surveillance sensitivity is shaped by the combined effects of larval diffusion, copepod density, and pond size, with interactions among these factors producing nonlinear relationships. Detection, in our model, was concentrated within a relatively restricted period after larvae matured to the infective stage and before dispersal and mortality reduced presence, indicating a limited spatiotemporal window for effective sampling. Surveillance performance peaked under intermediate dispersal regimes that generated sufficient spatial overlap between larvae and intermediate hosts, while both limited dispersal and excessive diffusion reduced detection by constraining encounters or diluting larval concentrations. Increasing habitat size reduced detection by diluting larval concentrations, but the magnitude of this effect depended on copepod density and dispersal dynamics, producing nonlinear and threshold responses rather than simple scaling with pond volume. Spatial and temporal patterns of detection shifted as larvae dispersed, with the most favorable detection periods occurring when both larval abundance and intermediate host encounters were elevated. These findings indicate that surveillance can be guided by local ecological conditions. When the timing of larval introduction is uncertain, effective surveillance requires repeated sampling over time to capture transient windows of detectability and the sampling will be less effective in very stagnant and highly mixed waterbodies. Overall, this study demonstrates how mechanistic modeling can support the design and interpretation of environmental surveillance strategies for Guinea worm eradication programs. Author summaryGuinea worm disease is close to eradication but confirming that transmission has fully stopped remains difficult because detecting infectious larvae in water is challenging. Transmission depends on freshwater copepods that become infected after ingesting Guinea worm larvae. These copepods are short-lived and unevenly distributed within ponds, and infected individuals may die before larvae reach the infective stage. As a result, environmental detection is inherently uncertain. We developed a three-dimensional agent-based model to simulate larval dispersal, copepod infection, and water sampling in a pond environment. The model shows that detection is constrained to a brief period when mature larvae and copepods overlap in space and time, and that this window depends strongly on local ecological conditions such as larval dispersal, copepod density, and pond size. Because infected copepods can be present outside these narrow detection windows, negative water samples do not necessarily indicate absence of transmission, highlighting the need for repeated, spatially targeted surveillance during the final stages of eradication.

Wildlife vaccination could become a powerful strategy to mitigate disease-induced biodiversity losses, yet many vaccines for wildlife diseases provide only limited protection. Notably, tools to control the fungal pathogen Batrachochytrium dendrobatidis (Bd) are urgently needed for amphibian conservation. Laboratory experiments have demonstrated that prophylactic exposure to Bd metabolites increases host resistance, significantly reducing infection intensity in amphibians subsequently challenged with live Bd. Because Bd metabolites are non-infectious and applied topically, this treatment has potential to be administered to waterbodies to vaccinate and protect amphibians. We developed an agent-based model that indicated imperfect vaccination could reduce or amplify Bd infections at the population level, depending on degree of enhanced resistance or tolerance. Utilizing a Before-After-Control-Impact design with ten years of data, we conducted an ecosystem-level trial where we applied low levels of Bd metabolites or a sham control treatment to ponds in California and subsequently quantified Bd prevalence and infection intensity in metamorphosing Pacific chorus frogs (Pseudacris regilla). Unexpectedly, infection intensity was significantly greater in treated ponds relative to control ponds following metabolite addition. Additional model simulations indicated that this could occur via two mechanisms: (1) if treatment greatly increased tolerance alone or in combination with smaller increases in resistance, or (2) if a deleterious environmental interaction caused the treatment to increase susceptibility, rather than promote resistance. Future research is needed to determine whether tolerance or environmental factors drove heightened Bd infection intensities in this field trial to identify contexts in which this treatment can be used as a conservation tool. Significance statementAlthough wildlife vaccination is increasingly explored as a strategy to mitigate disease-induced population declines, many available vaccines provide limited protection, requiring careful consideration to design successful conservation campaigns. Here, we use both an eco-epidemiological model and field manipulation experiment to assess the effectiveness of an imperfect prophylactic treatment (akin to a prototype vaccine) for chytridiomycosis, a disease implicated in the massive decline of amphibian biodiversity worldwide. We unexpectedly found that prophylaxis-treated ponds had higher pathogen loads relative to control populations and models suggest this could result from enhanced tolerance or an adverse environmental interaction.

Urban pollinator gardens can provide refugia and support diverse populations of native bees amid threats from habitat destruction, pesticides, and potential ecological pressures from the introduced honey bee (Apis mellifera (Linnaeus, 1748)). The University of California, Berkeley, maintained a native bee garden at the Oxford Tract research facility to study the biodiversity, phenology, and foraging habits of urban bees from 2003 to 2009. That garden was decommissioned, and a new garden was re-established in 2019. Using diversity observations from the early 2000s garden and non-lethal sampling techniques, we characterized plant-pollinator interactions between flowers and urban bees in the newer bee garden with a bipartite interaction network. Across 12 flower species, we observed two non-native pollinators, the honey bee (A. mellifera) and the alfalfa leafcutter (Megachile rotundata (Fabricius, 1793)), along with at least ten native bee species across three families (Apidae, Halictidae, Megachilidae). We found that, despite the garden being created for native bees, honey bees accounted for 84% of all pollination interactions. The most abundant native bees were sweat bees (Family: Halictidae). Generalist interactions dominated the network, as both honey and sweat bees foraged on most available flowers. Honey bees showed a significant positive correlation with floral abundance, visiting flowers with the highest number of inflorescences, whereas native bees did not show this preference. These results indicate that native bee garden stewardship could benefit from greater floral diversity, while avoiding the dominance of any single species with high floral abundance, thereby reducing the likelihood of direct competition with honey bees.

O_LIThe frequency and intensity of heat events is increasing across marine and terrestrial ecosystems. Within the same ecological community, the relative exposure and sensitivity to heat stress may vary considerably among interacting species, like predators and prey. This can be especially true for species that interact at the aquatic-terrestrial interface, as well as for interactions between primarily nocturnal and diurnal species, making it difficult to predict how such communities will respond to habitat warming. C_LIO_LIThermal limit metrics such as CTmax are often assumed to equate with ecological death because such temperatures impair behavioral activity and/or physiological functioning. Prey that are diurnally active can be more frequently exposed to temperatures that approach CTmax compared to their nocturnal predators, which may use thermal refuges during the day. Yet the impacts of daytime heat exposure on nighttime predation risk remain unknown. C_LIO_LIHere, we compared the thermal environment, performance, and heat tolerance between the predatory blue crab, Callinectus sapidus and one of its prey species, the mangrove periwinkle Littoraria anguilifera in a tropical mangrove ecosystem. We examined how exposing prey to heat stress at and below their CTmax affected their capacity to avoid predation in the field at night when predation risk is highest. C_LIO_LIWe found that acute exposure to temperatures near CTmax during the day increased the prey species susceptibility to predation during recovery at night. Although both interacting predator and prey have high thermal tolerance, prey are exposed to conditions that already reach CTmax, suggesting that current extremes in temperatures may already be influencing vulnerability to predation in this ecosystem. C_LIO_LIOur results suggest that differential exposure to sublethal heat stress in diurnal prey relative to their predator, along with the subsequent impact of these exposures on predation risk, will play a role in shaping these interacting as climate warms. C_LI

Interactions between cleaner fish Labroides dimidiatus and client fish, from which cleaners remove ectoparasites and mucus, represent a textbook example of mutualism involving sophisticated strategic decision-making. However, cleaners must also face intraspecific social challenges within a size-based hierarchy, where the largest females may eventually change sex and become males with higher reproductive rates. Following 540 individuals over 11 months, we found that, contrary to expectations, slow-growing females spent more time cleaning and cheated more frequently, without causing more negative client responses than fast-growing females did. Instead, variation in growth was best explained by social factors: fast-growing individuals experienced reduced social control, while slow growers spent more time in proximity to dominant individuals. As there was no evidence that spawning activity affected growth patterns, it appears that fast growth as a viable strategy for becoming a male largely depends on the lack of control by dominants.

A hosts diet can alter the course of parasite infection. This is especially true of trophic parasites, which a host acquires through feeding. While a large body of work attests to the role of diet in the spread of disease within-hosts, diet can also impact host density and encounter rate with parasites, both of which are expected to modify disease dynamics. When parasites are acquired through feeding, epidemics may be larger and more severe on high-quality diets if these diets support a higher density of hosts that feed more and thus ingest more parasites. Alternately, epidemics may be more severe on low-quality diets if malnourishment decreases hosts ability to resist disease. To differentiate these hypothesized effects of diet on disease, we quantified individual infections and epidemic dynamics for the natural microsporidian parasite Nematocida ironsii infecting its nematode host Caenorhabditis elegans. We measured feeding rate, parasite transmission, and host fitness across three bacterial diets that vary in quality and elicit distinct feeding behaviors in C. elegans. We found that low-quality diets reduced feeding rate, which corresponded to reduced acquisition of parasite spores. However, these diet-mediated differences in parasite acquisition did not directly map onto fitness consequences: hosts eating the poor-quality diet had similar reductions in fitness to those on higher quality diets. During epidemics, a combination of increased parasite acquisition and higher population growth rates resulted in higher parasite abundance for hosts on high-quality diets. Our work underscores the importance of considering both individual- and population-level impacts acting in concert to determine how diet affects the spread of infectious disease.

AO_SCPLOWBSTRACTC_SCPLOWMuscle efficiency decreases with increasing size, largely due to a relative decrease in its mechanical output. Muscle mechanical output depends on its activation, strain, and strain rate and thus varies between different muscles within a limb during locomotion. Distinct muscle coordination patterns are required for efficient cycling, and so we would expect that the coordination patterns for efficient cycling or indeed locomotion would change across animal sizes. We tested whether muscle coordination would change with muscle size using data derived from human cycling: this paradigm allowed for controlled changes in both crank torque and cadence, allowing the multifactorial problem of muscle power output to be decomposed. We used kinematic and pedal data from 12 cyclists undergoing steady pedalling at cadences from 80 to 140 r.p.m. and generated musculoskeletal simulations of their movements. We introduced novel multisegment muscle models in the simulation that incorporated the internal muscle mass and thus accounted for the scaling effects of muscle tissue inertia. We solved the simulations for the muscle activity that was required to minimise the metabolic cost during cycling for each condition. The masses of the muscle models were scaled across five orders of magnitude. The predicted muscle activations were classified by Principal Component analysis to identify whether the coordination of muscle activity was modulated across models with different sized muscles. Analysis of variance revealed significant changes in coordination at the large-scale factors. This study shows how the coordination of muscle activity during locomotion will likely change across a range of body sizes due to the non-linear effects of the inertial mass within the muscle tissues.

Adaptation to local environments enables species to thrive in diverse and challenging habitats. Steep elevational gradients provide a compelling natural adaptation laboratory, because abiotic conditions change progressively over short geographical differences. Given that elevation can strongly reshape physiology and behavior of insects, neuromodulatory systems offer a promising lens through which to examine elevation-specific adaptation. We challenged the hypothesis that adaptation to elevation involves octopaminergic signaling in honey bees (Apis mellifera), an important pollinator species occupying different elevations along East African mountains. We collected foragers from two distinct elevations at Mount Kenya (1,150 m and 1,900 m above sea level) and analyzed elevation-dependent changes in octopaminergic signaling. Tissue-specific analysis revealed a striking upregulation of all three octopamine {beta} receptor genes in the thoracic flight muscles and elevated octopamine brain concentrations at high elevation. Expression differences in the brain and fat body were rather modest. We subjected CRISPR/Cas9-mediated octopamine {beta}2 receptor knockouts to cold stress to study the function of octopaminergic signaling in thermoregulation. Loss of AmOAR{beta}2 reduced both the slope and amplitude of heating phases, indicating altered thermogenic dynamics. Together, these results identify the octopaminergic system as a central neuromodulatory regulator of thermogenic performance across elevations in honey bees. More broadly, our study highlights how modulation of conserved aminergic signaling pathways can shape physiological resilience to environmental gradients, pointing to a general mechanism by which insects adapt to changing thermal landscapes. Highlights- Bees from high and low elevation differ in expression of octopamine {beta} receptor genes and octopamine brain concentrations - CRISPR/Cas9-mediated octopamine receptor knockout alters thermogenic behavior - Octopaminergic signaling emerges as a key neuromodulator in thermal adaptation to elevation in honey bees Significance statementAnimals living along mountain gradients must cope with rapidly changing temperatures, yet the mechanisms enabling this adaptation remain poorly understood. We show that honey bees from higher elevations have increased brain octopamine levels and enhanced expression of octopamine receptors in heat-producing flight muscles. Using gene editing, we demonstrate that disrupting one key receptor alters how bees generate heat under cold stress. These findings identify octopamine signaling as a central regulator of thermogenesis and reveal a mechanism by which insects adjust to colder environments. More broadly, our results highlight how conserved neuromodulatory systems can fine-tune physiological performance, offering insight into how insects may respond to changing climates and expanding environmental extremes.

One of our sensory systems key functions is to detect threats in the environment. Sensory information eliciting negative emotions, such as fear or disgust, triggers instinctive avoidance reactions. This core survival mechanism is believed to be expressed as subtle non-conscious postural reactions, even when participants are instructed to stand still. Such avoidance behavior has mainly been studied using indirect measures that make participants aware of their posture (e.g. force-plate based methods) or measures that depend on explicit cognitive tasks, like moving a joystick to indicate an urge to approach or avoid the stimulus; experimental tasks with limited ecological validity and generalizability. Therefore, despite the importance of this basic survival strategy, its underlying mechanisms are still poorly understood. Here, we used a novel 3D-camera-based method allowing direct but implicit measures of postural reactions with high precision. Participants are aware that they are being filmed but, crucially, are not informed that distance measures are obtained. We assessed this ecologically valid measure of approach/avoidance responses in two different sensory modalities: olfaction and vision. Participants were standing upright while exposed to either olfactory or visual stimuli and verbally rating their perceived valence in each trial. In response to subjectively unpleasant odors and images, participants moved away from the stimulus source, as compared to pleasant stimuli. These results demonstrate a putative modality-independent early proxy for avoidance behavior in response to perceived negative valence. Considering its face validity and general applicability, this novel experimental method presents new possibilities for assessing non-conscious approach-avoidance responses in humans.

A central challenge in characterizing species niches is ensuring that foraging data accurately capture both the resources used and their relative importance, and the role of resource abundance in shaping foraging patterns. Most studies infer diet breadth and resource-use patterns from observational records, yet such data can mask resource-specific decisions when animals forage with different goals. Here, we test this experimentally using individually identifiable bees in controlled resource communities to quantify foraging decisions between nectar (for sustenance) and pollen (for offspring provisioning). Combining observations, pollen DNA metabarcoding, and pollen microscopy, we show that observed visitation patterns misrepresent the floral resources most important for offspring provisioning, which ultimately determines offspring survival and population persistence. We further show that interaction patterns are structured from processes beyond resource abundance. Our results demonstrate that commonly used observational approaches can mischaracterize diet breadth, potentially challenging conclusions about species generalization.

Behaviour is a key yet often overlooked component of animal camouflage and how it evolves alongside colour and morphology remains poorly understood. The repeated evolution of stick-like postures in spiders offers a useful framework for investigating the importance of behaviour for concealment, since matching the environment should rely on specific body forms and postures, not just colouration. We hypothesised that when spiders behaviourally align their body with the background orientation it should influence the shape, posture and colouration that best enhances camouflage. To test this, we used a genetic algorithm and human observers to evolve digital spiders to be harder to find. We evaluated how selection under three behavioural orientation treatments (aligned, random, and evolvable orientation) influenced spider capture time, background match (lightness and colour), posture, and body (cephalothorax and abdomen) dimensions. We found that spiders that behaviourally aligned with the background took substantially longer to find through evolving a better background match, and a more elongated posture and body shape than randomly orientated spiders. Our spiders mirrored the shape and posture adopted by numerous clades, illustrating how behavioural camouflage represents a key concealment strategy in structurally complex habitats, part of an interacting suite of traits that encompass successful concealment.

Social decisions often require animals to integrate information across multiple attributes of potential partners. Using biological motion stimuli, point-displays generated from tracked live shoals, we tested how adult zebrafish (Danio rerio) weigh shoal size and movement speed during social preference, and whether these preferences are susceptible to contextual manipulation by an asymmetrically placed alternative. In Experiment 1, we established a multi-attribute indifference point by presenting males and females with dichotomous contrasts in which shoal size and movement speed were traded off. Both sexes showed no preference when a larger, slower shoal (4 fish at 0.75x speed) was pitted against a smaller, faster shoal (2 fish at 1.25x speed), but preferred the smaller, faster shoal when the speed difference was greater (4 fish at 0.5x versus 2 fish at 1.25x), indicating that zebrafish are sensitive to graded differences in movement speed relative to numerical cues. In Experiment 2, unidimensional tests confirmed that both sexes preferred larger shoals when speed was held constant but revealed sex-based differences in speed sensitivity: males preferred faster-moving shoals at both shoal sizes tested, whereas females showed no significant speed preference. Male shoal size preferences were stronger at higher movement speeds, suggesting that speed modulates the strength of size preference. In Experiment 3, we tested the asymmetric dominance effect in males, the only sex sensitive to both dimensions, using the indifferent contrast from Experiment 1 as the primary options and four decoy shoals asymmetrically placed along either the size or speed dimension, under counterbalanced presentation orders. No decoy shifted male preference significantly from chance under any condition. These results indicate that zebrafish weigh social cues in a sex-specific manner, and that asymmetric decoy options do not induce preference biases in males when shoals vary along the dimensions of movement speed and size.

Many marine invertebrates are characterized by broad and highly plastic thermal limits, though the dynamic molecular mechanisms that enable extended thermal acclimation remain poorly understood. A classic example is the green crab (Carcinus maenas), which is a prolific and damaging non-indigenous species. Using a 22-day thermal exposure to cold (5{degrees}C), ambient (13{degrees}C), or warm (30{degrees}C) temperatures, we characterized plastic shifts in C. maenas performance using respirometry and time-to-right. We then used untargeted metabolomics and lipidomics analysis of heart tissues from days 4 and 22 to identify the molecular mechanisms underpinning plastic responses over time. Crabs at 30{degrees}C exhibited higher oxygen consumption rates than counterparts at 5{degrees}C. Interestingly, oxygen consumption rate increased over time at both temperatures, indicating thermal plasticity of aerobic respiration. Temperature-dependent metabolic reprogramming was employed by crabs to sustain aerobic respiration across temperature. Catabolism of branched-chain amino acids was important for energy production at elevated temperatures, while catabolism of arginine may have sustained the minimal energy needs of crabs exhibiting metabolic depression at cold temperatures. Righting response was positively correlated with temperature, and did not exhibit any changes over time. Lipidome remodeling consistent with homeoviscous adaptation could have enabled motor activity across temperature. Higher abundances of saturated and monounsaturated lipids likely provided structural integrity to cell membranes at 30{degrees}C, while lower abundances of these compounds may have enabled membrane fluidity at 5{degrees}C. Our work demonstrates the importance of ongoing molecular reprogramming in long-term acclimation, even when whole-animal physiology remains relatively stable. Summary StatementThis study demonstrates how the highly invasive green crab regulates metabolite and lipid pathways over time to maintain physiological performance across different temperatures.

Sex ratio is a key demographic parameter shaping population dynamics and evolutionary trajectories. In biocontrol agents, demographic bottlenecks during species introduction to a new habitat and subsequent mass rearing can elevate inbreeding, potentially biasing sex ratios through sex-specific mortality associated with inbreeding depression. Moreover, reproductive endosymbionts such as Wolbachia are known to manipulate host reproduction and further skew sex ratios. However, the relative contributions of these processes to sex-ratio variation remain poorly resolved. In this study, we evaluated the effects of cross-generational full-sibling inbreeding and Wolbachia infection on sex ratio and key life-history traits in the biocontrol beetle Zygogramma bicolorata using controlled laboratory crosses across three generations. Inbreeding did not significantly alter offspring sex ratio, which remained close to parity across generations, while pupal mortality increased in later generations, consistent with delayed expression of inbreeding depression. Adult body weight remained largely unaffected by inbreeding. Wolbachia infection was detected in a subset of females and was associated with a modest but significant increase in female-biased offspring production, although the effect was variable across lineages. Strain typing identified a single supergroup A Wolbachia, consistent with previous descriptions of the wBic strain from this species. These findings indicate that sex-ratio variation in introduced populations of Z. bicolorata is not driven by inbreeding alone but instead emerges from the interaction between demographic processes and symbiont-mediated effects, providing crucial insights for optimizing biocontrol programs where sex-ratio stability is essential for population establishment and persistence. SignificanceSex ratio is a key determinant of population growth and stability - the essential parameters determining success of biocontrol programs. Yet, the mechanisms shaping sex-ratio variation remain poorly resolved. Using controlled crosses in Zygogramma bicolorata, we show that short-term inbreeding does not directly alter sex allocation, despite inducing delayed fitness costs through increased pupal mortality. In contrast, Wolbachia infection contributes to female-biased offspring production, although with variable outcome across lineages. These findings demonstrate that sex-ratio variation in Z. bicolorata arises from the interaction of demographic processes and symbiont effects, rather than a single mechanism, with important implications for predicting the establishment, persistence, and efficacy of mass-reared biocontrol populations.