The juvenile hormone (JH) signaling pathway is crucial for reproductive development, molting, and metamorphosis in model insects. However, the molecular mechanisms of key components in the JH signaling pathway remain poorly understood in crustaceans. This study elucidates the function of RXR gene and JH analogs (JHAs) in the reproductive development of the brine shrimp Artemia parthenogenetica (A. parthenogenetica). Using an ELISA kit, we detected JH-like immunoreactivity in A. parthenogenetica that peaks during stage IV of ovarian development, coinciding with maximal expression of CYP15. The RXR homolog in A. parthenogenetica (Ar-RXR) features conserved DNA- and ligand-binding domains and shares close phylogenetic affinity with Daphnia magna (D. magna). Its expression profile aligns with JHA fluctuations, suggesting coordinated regulation of oogenesis. RNAi-mediated knockdown of Ar-RXR significantly suppressed RXR expression (P < 0.01) and downregulated JH pathway genes (Ar-Met, Ar-Kr-h1, Ar-Vg), accompanied by abnormal cytological characteristics: concentric membranes and lipid droplet accumulation in fat body cells, reduced yolk granules in embryos, and decreased offspring formation. Conversely, JH Ⅲ injection upregulated ovarian Ar-RXR and other JH pathway genes (except Ar-Kr-h1), yielding plump, well-arranged eggs. Co-injection of JH Ⅲ and dsRXR partially rescued JH pathway gene expression and reduced the number of egg abnormalities. Our findings demonstrate that a JH-like signal and Ar-RXR functionally interact to regulate reproductive development in A. parthenogenetica, providing mechanistic insights into JH signaling and advancing understanding of arthropod reproductive evolution.

Temporary adhesion by echinoderm tube feet is essential for attachment, locomotion and feeding, yet its molecular basis remains poorly understood in Holothuroidea. Here, we combined ultrastructural, proteomic, phylogenetic and expression analyses to investigate the temporary adhesive system in the sea cucumber Apostichopus japonicus. Scanning and transmission electron microscopy revealed a highly specialized tube foot disc epidermis featuring abundant secretory pores, dense microvilli, and two distinct types of adhesive secretory cells, whereas these features were absent in the non-adhesive homologous papillae. Proteomic analysis of tube foot footprints and discs, combined with bioinformatic screening, identified 20 high-confidence candidate adhesive proteins and one Astacin-domain-containing candidate de-adhesive protein. Notably, the 4,085-aa multidomain protein Aj-Sfp2-like was relatively abundant in the footprint proteome and showed extensive predicted post-translational modification. Tissue expression and in situ localization confirmed that Aj-Sfp2-like transcripts are localized to the secretory epidermis of adhesive appendages (tube feet and tentacles), whereas no such expression was detected in the non-adhesive papillae. Phylogenetic and comparative domain analyses demonstrate that Aj-Sfp2-like belongs to the broadly conserved Sfp2-like protein family, sharing a homologous modular framework with asteroid-restricted Sfp1-like proteins. These findings provide the first molecular framework for understanding tube foot adhesion and de-adhesion in holothurians and broaden our understanding of the conserved and divergent basis of temporary adhesion across echinoderms.

Skeletal muscle is reliant on the myosin ATPase enzyme to power locomotion, making muscle and locomotor performance thermally sensitive. However, muscles performance is often enhanced by elastic tendons, and the power amplification they enable is thought to increase sprint performance and its thermal robustness. The previous study suggests in vivo ankle muscle powers that exceed predicted muscle powers and may contribute to the high thermal robustness of sprint performance in S. occidentalis. Here, we directly measure gastrocnemius muscle power and its thermal sensitivity, compare this to in vivo ankle muscle power to quantify power amplification, and determine the potential for the gastrocnemius muscle-tendon unit (MTU) to store the required elastic strain energy. Peak gastrocnemius muscle power was ∼83, 156 and 371 W kg-1 at 15, 25 and 35°C with Q10 values of ∼1.9-2.4. Peak in vivo muscle power at the ankle was ∼180, 622 and 881 W kg-1, indicating a ∼2.2-, 4.1- and 2.4-fold power amplification, at 15, 25 and 35°C, respectively. The fixed-end compliance of the gastrocnemius was ∼17%, series elastic elements experienced an ∼12% strain, and the muscle fibers stored ∼17 J kg-1. Hence, this species clearly uses power amplification in sprinting at all temperatures, and the high degree of power amplification at 25°C probably increases thermal robustness of sprint performance at 25-35°C. The degree of elastic strain energy storage observed in this MTU is comparable with avian, mammalian and anuran MTUs known to store and utilize elastic strain energy and modestly amplify muscle power.

The maintenance of locomotor performance across temperatures is critical to ectotherm fitness but is limited by the thermal sensitivity of the myosin ATPase enzyme. However, sprint performance in lizards appears more thermally robust than the muscles powering it, potentially due to power amplification by elastic tendons. To explore this, we determined the thermal sensitivity of Sceloporus occidentalis acceleration at 15-35°C, used inverse dynamics to estimate in vivo muscle powers at hindlimb joints and compared these to predicted maximum muscle powers. The Q10 of acceleration was 1.7-2.5, compared with literature Q10 values of 2-3 for muscle power. Maximum in vivo power was ∼180, 622 and 881 W kg-1 at the ankle, ∼127, 393 and 433 W kg-1 at the knee, and ∼41, 176 and 193 W kg-1 at the hip at 15°C, 25°C and 35°C, respectively. Comparison with predicted maximum muscle powers suggest amplifications of ∼2.2, 3.8 and 2.7-fold at 15°C, 25°C and 35°C at the ankle. There was little power amplification at the knee and hip except for a ∼2.4-fold amplification at the knee at 25°C; hip power appeared sub-maximal at 35°C. This is consistent with the limited presence of in-series elastic elements at the knee and, particularly, the hip. These results suggest that power amplification at the ankle is critical to the sprint performance of S. occidentalis and its thermal robustness of 25-35°C, but that there is an additional limit to sprint performance at 35°C that may contribute to the apparent thermal robustness.

Environmental conditions during embryonic development wield critical influence on organismal traits. For example, in species with temperature-dependent sex determination (TSD), the incubation temperatures an embryo experiences determines sex. Reaction norms quantifying this relationship are essential tools for predicting how climatic variability will shift population sex ratios. However, existing TSD reaction norms are derived from constant temperature laboratory conditions, leaving open the question of their applicability to natural conditions where incubation temperatures fluctuate. In this study, we developed a TSD reaction norm for the American alligator (Alligator mississippiensis) using fluctuating temperature treatments. We incubated eggs under five fluctuating temperature regimes, determined offspring sex using gonadal gene expression, and used the resulting sex ratios to fit reaction norms based on mean temperature and constant temperature equivalents (CTE). The reaction norms followed the canonical Female-Male-Female pattern of crocodilians. At lower incubation temperatures, our fluctuating-temperature reaction norms closely aligned with that derived from constant temperatures, suggesting that mean temperature and CTE statistics perform reasonably well in this range. However, at higher incubation temperatures, the range most relevant to climate warming projections, our reaction norms diverged substantially from constant temperature reaction norms with upper pivotal temperature estimates differing by up to 1.36°C. Because small thermal shifts at this critical transition can dramatically alter predicted sex ratios, these discrepancies carry real consequences for conservation forecasting. Our results underscore the importance of incorporating ecologically realistic thermal variation into TSD reaction norms and highlight the upper male-to-female transition as a priority for mechanistic study.

Hypoxia in plant tissue should affect animals living within them. Gall makers stimulate their plant hosts to produce the gall they inhabit and feed on, and also influence the gall phenotype for other adaptations, such as defence against predators. In the context of adapting to potential gall hypoxia through the extended phenotype of the gall, we studied the prevalence and conditions leading to hypoxia in goldenrod galls induced by Eurosta solidaginis. Larval respirometry data showed only mild tolerance to hypoxia, based on elevated critical oxygen partial pressures (∼10 kPa). Mathematical modelling of gas diffusion into the gall suggested that the extent of prevalent hypoxia is tolerable to the larva, while more severe temporary hypoxia may occur early in the host plant's growth season. As galls mature, the cortex layer that makes up most of the gall volume becomes less metabolically active and more permeable to gas diffusion, which is important in preventing severe hypoxia. While the mature cortex layer is unlikely to have been specifically an adaptation to hypoxia, it could mitigate the need for other trade-offs in gas exchange. Since gall makers can influence gall size and tissue composition, this raises the possibility that, on top of the already established predation trade-offs, the gall maker must balance the hypoxia risk of remaining in less differentiated, cytoplasm-rich tissue with the costs associated with inducing a larger, more mature gall with greater gas diffusion capacity.

Biomineralization evolved repeatedly across animals, resulting in novel strategies for buoyancy, locomotion, and defense. Sponges are the only metazoans that build silica-based skeletons, and the mechanisms underlying their biosilicification are relatively understudied compared to other major silicifiers. Here, we show that in freshwater sponges, cells specialized for biosilicification, termed sclerocytes, highly express lysosomal-associated genes, including the V-H+-ATPase (VHA) proton pump, oculocutaneous albinism type 2 (OCA2) anion channels, cathepsins, silicateins, TMEM55A/B, TMEM192, TMEM199, and other proteins involved in lysosomal function. We demonstrate that VHA protein localizes to the sclerocyte and propose that VHA-dependent silica deposition vesicle (SDV) acidification is likely essential for silica spicule formation. This function mirrors the role of VHA in diatom biosilicification and in biocalcification across a broad sweep of taxa. We also found that genes homologous to the plant silicon efflux transporter Lsi2 are highly upregulated in sclerocytes and confirmed their localization using Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH). These results corroborate phylogenetic and transcriptomic evidence that Lsi2 homologs are involved in sponge silicic acid transport. Data mining of previous studies revealed that many of the sponge sclerocyte lysosomal-associated genes have been correlated with biomineralization across eukaryotes, including coccolithophores, bryozoans, crustaceans, mollusks, and mammals. As detoxification was a likely driver for the evolution of biomineralization, we propose that lysosomal machinery aided in detoxification by allowing sponges to sequester Si in acidified compartments to prevent its catastrophic intracellular precipitation. This lysosomal toolkit was likely independently co-opted for the formation of biomineralized structures in multiple species.

The field of aquatic toxicology has undergone a paradigm shift in recent decades. The movement away from observational data detailing the concentrations that produce deleterious effects and towards a mechanistic understanding of how toxicants affect organismal physiology has significant advantages for the field. This mechanistic approach allows for the prediction of toxicant effects across species and among toxicants with similar modes of action. Critically, knowledge of the pathways by which toxicants exert their impacts can provide insight into how environmental factors such as temperature, salinity, dissolved oxygen, pH, alkalinity, dissolved organic carbon, major ions and co-occurring toxicants interact to modify toxicity, providing a more nuanced understanding of toxicity in natural settings. This mechanism-based approach has driven changes in the regulatory environment, through the incorporation of predictive modelling tools that can determine, on a site-specific basis, safe thresholds for concentrations of toxicants in aquatic settings. This represents an exemplar of how fundamental experimental biology translates into real-world application. However, we argue that while mechanistic aquatic toxicology is an important subfield of experimental biology, there are a number of factors that make it unique. In this Commentary, we outline best practices for the experimental biologist conducting research into toxicant effects on aquatic biota, provide examples of the utility of mechanistic approaches to the field of aquatic toxicology and outline how future advances in aquatic toxicology could benefit from experimental biology approaches.

Most animals rely on flexible fuel-use strategies in which carbohydrates support short, high-power bursts of activity and fats fuel prolonged, low-intensity locomotion. A notable exception occurs in a small subset of taxa - primarily migratory birds, bats and some insects - that sustain vigorous, high-intensity aerobic locomotion while relying largely on lipid oxidation. In this Review, we examine why this extreme, fat-powered endurance phenotype has evolved so rarely. Synthesizing evidence across locomotor modes (swimming, running, flying), body sizes and patterns of endurance specialization, we argue that selection for endurance alone is insufficient to drive the evolution of this phenotype. Sustained fat-powered endurance emerges only where scaling constraints on power, heat dissipation and oxygen delivery are not prohibitive, the ecological demand for prolonged activity aligns with mechanical conditions that permit continuous high aerobic output, and the benefits of specialization outweigh the evolutionary costs of reduced metabolic flexibility. In most animals, endurance demands are intermittent or context dependent, favoring partial specialization that enhances aerobic capacity and lipid use while preserving the ability to shift fuels and performance modes. Crossing into this extreme phenotype requires coordinated, reversible co-expression of structural and regulatory changes that together commit organisms to a narrow performance phenotype and impose trade-offs in force production, metabolic versatility and resource allocation. Consequently, evolution often favors trajectories that approach, but do not fully cross into, this exceptional performance state. Together, these patterns show that sustained, high-intensity fat-powered endurance is a rare and conditional evolutionary outcome, arising only where ecological necessity, biomechanical permissibility and physiological feasibility align.

Human activities are rapidly altering marine ecosystems, with increasing vessel traffic elevating underwater sound levels worldwide. Polar regions are particularly vulnerable, as declining sea ice has expanded access to the Arctic Ocean. Arctic cod (Boreogadus saida) is a key species that spawns under sea ice, yet its hearing abilities remain entirely uncharacterised. Understanding how this species detects sound is essential for assessing the potential impacts of rising anthropogenic sound. Here, the detection sensitivity of spawning and non-spawning Arctic cod were evaluated using auditory evoked potentials (AEPs). Both groups were most sensitive to frequencies below 200 Hz when assessed using sound pressure. However, particle motion sensitivity differed markedly: spawning fish showed peak sensitivity at 400 Hz, whereas non-spawning fish were most sensitive near 90 Hz. Overall, Arctic cod exhibited significantly enhanced hearing sensitivity and an expanded detectable frequency range during spawning compared with non-spawning individuals. These results provide the first evidence of seasonal differences in auditory plasticity in a broadcast-spawning fish and highlight the potential vulnerability of Arctic cod spawning behaviour to increasing anthropogenic sound in a rapidly changing Arctic environment.

Despite a likely role in setting stress tolerance limits, food ration has received limited attention as an ecological factor affecting the cellular stress response (CSR). To study the interactive effects of food and temperature on the proteomic heat shock response, we acclimated intertidal mussels (Mytilus californianus) to four combinations of nearshore (low) and aquaculture (high) phytoplankton levels, combined with low (20 °C) and high (30 °C) aerial temperatures during daytime low tides. Mussels were then exposed to an acute (6 h), aerial heat stress (33 °C) and allowed to recover for 1 h and 25 h in pre-exposure conditions. Proteomic changes in the gill before and after heat shock were measured using label-free liquid-chromatography-mass spectrometry. Compared to other acclimation treatments, low-food-low-temperature (LTLF) mussels modified more splicing factors, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and translation initiation and elongation factors, suggesting systemic changes to RNA processing, selection, and translation. LTLF mussels also increased chaperones of the actin- and microtubule-associated cytoskeleton and proteins that mature along the endoplasmic reticulum to Golgi secretory pathway. Simultaneously, actin stress fiber formation at focal adhesions and the extracellular matrix, along with anchoring of microtubule-associated cilia, indicate a possible system-wide mechanical breakdown of the cytoskeleton. Signaling proteins causing cytoskeletal changes varied mainly in LTLF mussels and suggest a food-dependent role for prostaglandin synthesis. Overall, the acclimation-dependent proteomic changes show how thermal conditioning and food ration together will shape the CSR of mussels during heat waves.

Hydration state is an important regulator of vertebrate physiology, yet the temporal dynamics of hydration-associated immune responses remain poorly understood. We investigated how progressive dehydration and repeated dehydration-rehydration cycles influence innate immunity in Antaresia childreni (Children's pythons). Snakes were assigned to either a hydrated control group or a dehydration-treatment group, and plasma osmolality, agglutination, lytic ability, and bacterial killing ability were measured throughout two complementary experiments. Progressive dehydration produced stepwise increases in plasma osmolality, whereas immune responses changed later and differed among immune components. Agglutination and lytic ability increased during later stages of dehydration, whereas bacterial killing ability increased only at peak dehydration; following rehydration, no immune measure differed significantly between treatment and control animals. During repeated dehydration-rehydration cycles, plasma osmolality increased during both dehydration events and returned following rehydration, whereas immune responses differed in their persistence and repeatability. Agglutination remained higher than controls throughout both dehydration-rehydration cycles, lytic ability increased during both dehydration events but declined following rehydration, and bacterial killing ability increased during the first but not the second dehydration event. These findings demonstrate that innate immune function is influenced not only by dehydration, but also by the temporal dynamics and recent history of water limitation. More broadly, our results emphasize that physiological responses to environmental change cannot always be inferred from endpoint comparisons alone.

Magnetoreception - the ability to detect Earth's magnetic field - is widespread among animals. Despite recent progress, fundamental gaps in our knowledge remain. The two main goals of modern magnetoreception research are to: (1) understand how magnetic information is used by animals in navigation; and (2) uncover the biophysical mechanisms that underlie magnetic field detection. The discovery that animals use magnetic information not only as a directional (compass) cue, but also as a kind of 'map', has revolutionized research in animal navigation. Recent findings suggest that animals use magnetic cues flexibly and idiosyncratically to help guide spatial movements. We propose that a valuable but seldom used approach to studying animal movements is to consider ways in which seemingly complex navigational feats can be accomplished with minimal information, often without the need for classical map-and-compass navigation, a cognitive representation of the environment, or prior knowledge of the terrain. We also used a recently developed model to demonstrate that natural selection can potentially 'tune' magnetic navigation systems to optimize navigation in different parts of the world, and we illustrate the possibility with examples of long-distance migrations by sea turtles, fishes and birds. Emerging evidence further implies that there are multiple magnetoreceptive mechanisms rather than a single, universal way of sensing magnetic fields. We argue that the path forward, for both unraveling animal navigation and elucidating mechanisms of magnetoreception, requires a thoughtful balance between a search for unifying principles and an expectation of major differences among species and populations.

Glucocorticoids (GCs), such as cortisol and corticosterone, are hormones that have been widely linked to stress and thought to inhibit reproductive function. However, this conclusion is largely based on studies that administer exogenous GCs, rather than an understanding of the relationship between natural, endogenous GCs and reproduction. To begin to address this gap in our knowledge, this systematic review aims to characterize endogenous GC patterns across reproductive stages in a range of vertebrates. Specifically, we focused on GC patterns across the ovarian cycle in healthy, sexually mature females across vertebrate taxa. From more than 3000 papers screened, we identified 68 that met our inclusion criteria. We found that GCs show predictable fluctuations across the ovarian cycle, with 73% of the studies reporting a significant change in GCs across stages of the cycle. In the majority of studies, GCs were highest around the time of ovulation. This suggests that increases in GCs might promote or support certain aspects of reproductive function, in contrast to the prevailing dogma that GCs always inhibit reproduction. We discuss evidence that GC peaks around ovulation might stimulate oocyte maturation (either alone or synergistically with luteinizing hormone) and/or regulate inflammatory processes related to follicle rupture. Recognizing that increases in GCs might support aspects of ovarian function across taxa represents a paradigm shift in how we think about the relationship between GCs and reproduction, moving us towards a new realm for understanding reproduction and reproductive failure that, as of yet, remains relatively unexplored.

Microglia govern multiple aspects of brain architecture and function by eliminating dying neurons and glia, stimulating neurogenesis, refining neural connections, and orchestrating immune responses. The Mexican tetra, Astyanax mexicanus, is a powerful model system for investigating the evolution of brain function, yet microglia have not been investigated in this system. A. mexicanus exists as surface- and cave-dwelling morphotypes with prominent behavioral and physiological differences. Notably, these evolved behavioral and physiological changes in the cave morphotype, including diminished immune responses and alterations in sleep, circadian rhythms, and sensory processing, are directly linked to known microglial functions in other systems, suggesting that evolved differences in microglia may shape brain circuitry adaptations in cavefish. Here we develop an experimental toolbox to examine microglial specification, dynamics, and function in A. mexicanus and perform comparative analysis of microglial cell biology between the surface and cave morphotypes. We find that the cave populations show increased numbers of microglia over developmental time relative to their surface counterparts. Microglia in A. mexicanus exhibit a rapid expansion in response to inflammatory cues, distinct from the inflammatory response observed in the related teleost zebrafish. Furthermore, microglia in cave populations exhibit lower lysosomal pH and expanded proteolytic compartments relative to surface morphotypes. Together, our observations reveal evolved differences in microglial cell biology between surface and cave populations of A. mexicanus and provide a framework to uncover novel neuroimmune mechanisms underlying the remarkable adaptations of the Mexican tetra.