Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

In many oviparous animals, egg yolk is the sole source of nutrition until feeding begins, and carbohydrates are present in only small amounts in the yolk. Glucose plays an important role in the developmental processes of various animals. In addition, gluconeogenesis has been reported to occur in the yolk syncytial layer (YSL) of cartilaginous fish and teleosts. In contrast, the role of gluconeogenesis in tetrapods remains unclear. In this study, we used Xenopus tropicalis, an anuran amphibian, which lacks YSL, and therefore provide an opportunity to examine the evolutionary conservation of gluconeogenic mechanisms among vertebrates. In X. tropicalis, liquid chromatography/mass spectrometry revealed that glucose levels increased before liver formation. Subsequent tracer experiments using 13C-labeled metabolic substrates detected gluconeogenesis activity from glycerol and lactate. Expression analyses showed that gluconeogenic genes are expressed in the epidermis and endoderm. Consistently, G0 knockout of fbp1, a key gluconeogenic gene, resulted in a significant reduction in glucose levels, affecting brain development. These findings first demonstrate that gluconeogenesis supports development of X. tropicalis. To the best of our knowledge, gluconeogenesis in developing epidermis has not been reported, highlighting previously unrecognized diversity in tissue-specific metabolism during vertebrate development. Comparative analyses across species will provide further insights into the evolution and functional significance of embryonic gluconeogenesis and nutrient metabolism.

The presence of multiple discrete color patterns within a species has captivated evolutionary biologists for more than a century, especially when such polymorphism is confined to one sex. The brown anole Anolis sagrei exhibits a female-limited polymorphism in dorsal patterning, which is controlled by allelic variation at the autosomal gene CCDC170. Here, we present and test a threshold model that can explain why the polymorphism is female-limited. We hypothesize that allelic variation at the CCDC170 locus affects only female color pattern because this gene is co-expressed with its neighboring gene ESR1, highly expressed in female, but not male, embryos. By manipulating embryonic estradiol levels, we show that genetic males can be induced to express the polymorphism according to allelic variation at the CCDC170 locus, which is naturally masked by low expression levels of this gene. Inversely, treating genetic females with fadrozole, which depletes estradiol, leads to monomorphic patterns irrespective of genotype, as for natural males. Using RT-qPCR, we demonstrate that these effects are accompanied by a direct influence of estradiol and fadrozole on gene expression levels of CCDC170 and ESR1, thereby validating the threshold model. Our results suggest that the CCDC170-ESR1-locus is part of a mechanistic link between the morph-determining and the sex differentiation systems and provide a causal explanation for the developmental origin of a sex-limited color polymorphism.

At a mere 20 cells, the Caenorhabditis elegans intestine regulates metabolism, energy homeostasis, host defense, yolk production, and genetic aging, all while dynamically responding to its environment. How the intestine develops to carry out these disparate functions is unknown, and how cells differ along the length of the intestine is unclear. To address these questions, we performed single-cell RNA sequencing (scRNA-seq) on FACS-enriched intestinal cells from mixed-stage C. elegans embryos. The resulting single-cell transcriptomes of 974 cells organized into 13 clusters, suggesting a diversity of cell types and states. We used two post hoc approaches to ascribe identities to each cluster. First, genes with known developmental timing in early-, mid-, and late-stages were used to place clusters in time, and smiFISH microscopy was used to fine-tune the assignments. Second, the eight late-stage clusters were assessed for their region of origin. To assign these clusters to anatomical regions, we identified marker genes for each cluster and assessed their expression along the anterior-to-posterior length of the intestine using smiFISH microscopy. Genes associated with growth and cell division were expressed in early stages, whereas genes associated with immune responses and metabolism were expressed later. Genes associated with biotic responses and RNA metabolism were the most likely to vary across the intestines anterior-posterior axis. Finally, perturbation of anterior-localized intestinal transcripts more robustly affected intestinal function compared to central or posterior-localized genes. Overall, this research illustrates the intrinsic heterogeneity across the 20 cells of the embryonic intestine and sets the stage for future works aimed at understanding cell-specific intestinal responses to diet and the environment. ARTICLE SUMMARYWe investigate how the Caenorhabditis elegans intestine develops specialized functions on a spatiotemporal scale. We used single-cell RNA-sequencing to analyze embryonic intestinal cells and identify 13 distinct clusters. Combining gene expression analysis with microscopy, we assigned clusters to developmental stages and anatomical regions. Clusters associated with early intestine development express genes linked to growth and cell division, while later-stage clusters express genes involved in metabolism and immune responses. Genes varied across the intestines anterior-to-posterior axis, and disrupting anterior-specific genes produced stronger functional effects. These findings reveal previously unrecognized intestinal diversity and provide insight into how intestinal cells specialize during development.

Acropora spp. are the dominant reef-builders of the Indo-Pacific but are also amongst the most stress-sensitive corals. For these reasons, Acropora spp. have become the most studied of corals, two species (A. digitifera and A. millepora) often essentially serving as the basis for understanding molecular responses and processes across the sub-order Refertina and corals in general. The early development of these species has been well-characterised in terms of morphology and gene expression but as yet we have a limited understanding of how transcription is regulated during development. In "higher" animals (bilaterians) microRNAs (miRNAs) are critical regulators of gene expression but until now their involvement in coral development has not been investigated. Building on the existing developmental data for Acropora spp., we catalogued microRNAs (miRNAs) expressed during the early development of Acropora digitifera and profiled their expression in 21 stages from unfertilised eggs to 24h after treatment with a natural settlement cue (CCA chips). 157 miRNAs were recognised, many of which ([~]60%) were novel. These fell into three distinct groups, corresponding to three distinct developmental phases: (1) those present in eggs through to gastrulation (2) a larvally expressed group and (3) those expressed following settlement induction. Exposure of competent larvae to a natural settlement inducer resulted in major changes in the miRNA profile within 10 minutes, indicating that miRNAs may be particularly important in mediating the larva/polyp transition but are also likely to play important regulatory roles throughout early coral development in addition to possible roles in disease resistance.

Understanding how anatomical structures evolve requires disentangling the roles of integration and modularity in shaping morphological variation. The vertebral column, a serially repeated and regionally differentiated structure, provides a powerful system for investigating these processes. Here, we examine how vertebral morphology evolves in relation to whole-body elongation across the adaptive radiation of Lake Malawi cichlid fishes. We tested for evolutionary integration between the precaudal and caudal domains, as well as assessed the contributions of vertebral count, centrum shape, and intervertebral spacing on body elongation. We find strong evolutionary integration between precaudal and caudal vertebral shape, with both vertebral shapes varying along shared axes of multivariate shape change. Despite this, precaudal and caudal vertebral counts evolve independently, indicating a decoupling between the evolution of identity and morphology. Whole-body elongation is significantly associated with coordinated changes in vertebral and rib morphology, including proportional increases in centrum size, posterior displacement of neural and haemal spines, and increased rib curvature. In contrast, centrum elongation and intervertebral spacing do not independently explain body elongation beyond vertebral counts. These results demonstrate that body elongation in cichlids necessitates integrated, multivariate changes in axial morphology. Our findings highlight the importance of morphological integration in facilitating coordinated evolutionary responses in anatomical systems.

In many tropical areas, seasonal rainfall leads to distinct dry and wet seasons. Many butterflies developing under wet season conditions develop into adults with large ventral eyespots on the wing margins, whereas those developing under dry season conditions have smaller or no eyespots. In greener, wet season habitats, larger eyespots can divert predator attacks toward the wing margins, while reduced eyespot size improves camouflage in the dry leaf litter-dominated habitat during the dry season. However, the dry season is also characterised by higher desiccation stress than the wet season. We hypothesised that larvae developing under dry season conditions develop into adults with higher desiccation tolerance than those reared under wet season conditions. We tested this by rearing larvae of the butterfly Mycalesis mineus under simulated dry and wet season conditions and assaying the desiccation tolerance of the resulting adults. Butterflies reared in dry conditions survived longer under desiccation stress, lost lesser water during pupal-adult metamorphosis, and were heavier than those reared in wet conditions. We also tested the correlation between eyespot size and desiccation tolerance. A negative correlation between the traits would be expected if similar developmental pathways regulate them. Consistent with this expectation, individuals with smaller eyespots had higher desiccation tolerance. Our results demonstrate plasticity in desiccation tolerance, and suggest that predator avoidance and desiccation tolerance traits may share similar developmental pathways.

Linking animal movements to environmental drivers is essential for understanding ecological processes and anticipating species responses to climate change. We investigated habitat-specific movements in a globally significant aggregation of loggerhead turtles (Caretta caretta) nesting in Cabo Verde. Satellite tags on 15 adults (12 females, 3 males) provided multi-year tracks spanning breeding, migration, and foraging habitats. Movements and phenology differed by habitat. During the breeding season, females used either coastal areas, remaining within [~]20 m depth, or undertook long looping forays up to 360 km. Males showed two strategies: two remained resident in Cabo Verde waters, including Fra, the largest male tracked (Curved carapace length of 105 cm compared with a male mean of 90.7 {+/-} 10.3 cm), while the third migrated annually to distant foraging grounds and returned ahead of the subsequent breeding season. In foraging habitats, turtles adopted neritic or oceanic strategies: neritic turtles remained localised in warm, productive waters, whereas oceanic turtles ranged widely in deeper, less productive areas. Time- and space-shift analyses showed that oceanic foragers used intermediate sea surface temperature and chlorophyll-a conditions relative to nearby or temporally shifted alternatives, consistent with movement within a thermal-trophic trade-off. Together, these results show how sex, body size, and energy balance drive habitat-specific movement dynamics in a changing ocean.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

Silk glands have been found in two groups of amphipods: the Corophiida and the Ampeliscidae. The silk glands in Ampeliscidae, however, have yet to be examined in detail. Here we report, for the first time, the morphology and distribution of pereopodal glands in the Ampeliscidae, in non-thread producing Synopiidae, and in the Paragammaropsidae. In the Ampeliscidae we found two gland types distributed throughout all pereopods which have the ability to create threads. Pereopods three and four have additional silk extrusion morphology at the tip of the dactylus in which silk is transformed into semi-cylindrical threads used for building domiciles. Synopiid outgroup species have one of the gland types but lack silk extrusion morphology. Using ancestral state reconstruction analysis, we find that glands in the Synopiidae are likely ancestral and hypothesize that silk glands in Ampeliscidae are derived from these ancestral glands. Silk-spinning pereopods in the Paragammaropsidae had similarities with both Corophiida and Ampeliscidae but had distinctions. Ampeliscidae silk-spinning systems bear surprising resemblance to the Corophiida which presents one to reconsider the taxonomic placement of Ampeliscidae and the origins of silk-spinning in amphipods. This is the first comprehensive study on the glandular systems of Ampeliscidae, Synopiidae, and Paragammaropsidae using advanced microscopy, providing pertinent morphological data to the study of arthropod silk gland evolution and complex traits.

Conspecific sperm precedence via cryptic female choice is a post-ejaculatory selection process that reduces hybridization, and can be pronounced in sympatric species. In their native Europe, Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) exert conspecific sperm precedence under heterospecific sperm competition, which is at least partially enabled by female reproductive fluid. We examined post-ejaculatory selection of both species in Newfoundland, Canada, where Atlantic salmon evolved in absence of brown trout, but now experience hybridization threats due to anthropogenic introductions. Using split-ejaculate and split-clutch in-vitro fertilizations we evaluated whether allopatric evolution has relaxed this selection in Atlantic salmon, and found that they had no ability to bias paternity towards conspecific males, whereas naturalized brown trout retained a strong ability to do so. Female reproductive fluid influenced this, as when fluid associated with a species eggs was swapped, hybridization increased. In the artificial situation of no female reproductive fluid during sperm competition, paternity changed dramatically, but sperm swimming performance did not predict it. Our findings contribute to understanding the evolution of cryptic female choice and how the mechanisms of reproductive isolation can be reinforced through sympatry, while also highlighting a new potential conservation concern for North American Atlantic salmon.

The diversity of body shapes is one of the most prominent features of phenotypic variation in mammals. Yet, mammalian body shapes are poorly quantified and the underlying components contributing to its diversity as well as its relationship to other components of the skeleton are rarely tested. Here, we use lagomorphs (hares, rabbits and pikas) as a model system to (1) investigate which components of the skeleton contributed the most to body shape diversity, (2) examine the relationships between body shape and relative limb lengths, and (3) test how body size, ecotype, burrowing behavior, and locomotor mode influenced variation in lagomorph body shape and appendicular morphology. We quantified the body shape and functional proxies of the appendicular skeleton in 40 lagomorph species from osteological specimens held at museum collections. Using phylogenetic comparative methods, we found the relative length of the ribs and elongation or shortening of the thoracic and lumbar regions contributed the most to body shape evolution across lagomorphs. Second, we found that only leporids (hares and rabbits) exhibited a significant relationship between limb length and body shape, where more elongate species exhibit relatively shorter forelimbs and hindlimbs. Lastly, we found that models incorporating body size were the best predictors of lagomorph body shape and the majority of the appendicular traits, whereas models incorporating burrowing behavior and locomotor mode were largely poor fits. Broadly, these results indicate that larger lagomorphs tend to exhibit more robust body shapes with longer, more gracile forelimbs, whereas smaller lagomorphs tend to exhibit more elongate body shapes with shorter, more robust forelimbs. Overall, this work contributes to the growing understanding of mammalian body shape evolution and demonstrates the importance of not omitting body size in ecomorphological analyses.

O_LITerrestrial insects are vulnerable to desiccation due to their small body size. Because insects lose most water through cuticular evaporation, cuticular traits strongly influence desiccation tolerance. Individuals with greater cuticular melanisation, i.e., darker ones, are hypothesised to tolerate desiccation better than less melanised ones. C_LIO_LIIn many butterflies, pupal colour is plastic - individuals pupating on leaves tend to be greener, while those that pupate away from leaves (off-leaf), such as on tree bark or defoliated twigs, tend to be browner. Brown pupae are hypothesised to have more cuticular melanin and are expected to experience higher desiccation stress than leaf-borne green pupae. Thus, plasticity in pupal melanisation may be an adaptation against desiccation. We tested this in the butterfly Eurema blanda. C_LIO_LIWe demonstrate that individuals pupating on on-leaf substrates are greener than those pupating on off-leaf substrates, and that desiccation stress is higher in the off-leaf substrates, a microenvironment typical of brown pupae, than in typical green pupae. Using Raman spectroscopy, we show that brown, but not green, pupal cuticles contain melanin. C_LIO_LIFollowing this, we obtained greener and browner pupae by manipulating substrate colour. When subjected to desiccation stress, browner pupae survived better than greener ones. There was no correlation between pupal colour and survival in the absence of desiccation stress. Thus, melanisation appears to confer a survival advantage to pupae by increasing desiccation tolerance. C_LIO_LISurvival under desiccating conditions was inversely related to water loss. Interestingly, melanisation did not correlate with water loss, suggesting that melanisation helps tolerate desiccation through physiological mechanisms not directly related to water loss reduction. C_LIO_LIOur findings reveal an additional, crucial, adaptive value of pupal colour plasticity, a trait that has been studied primarily from an anti-predatory perspective. C_LI

Arthropods exhibit an exceptional diversity of life histories, where developmental modes involve moulting stage progressions with changes ranging from the bare minimal to the dramatically transformative. While this variability drives many research questions aiming to understand evolutionary and developmental underpinnings of life history differences, it can complicate comparative analyses across taxa. However, this can be approached by applying a framework that defines metamorphosis as a post-embryonic stage progression characterised by substantial changes in morphology and adaptive landscape. Employing this framework with a phylogenomic dataset spanning 26 orders and encompassing four independently arising metamorphic lineages, we explore gene repertoire evolutionary dynamics potentially associated with metamorphosis in Pancrustacea. The approach contrasts gene family evolutionary dynamics inferred to have occurred in the last common ancestors of the metamorphic Insecta, Copepoda, Eucarida, and Thecostraca, with those of their sister lineages, as well as of descendent and ancestral nodes. The results reveal that the metamorphosis ancestors are characterised by an elevated number of gene family births and expansions. Expanded gene families share a set of commonly enriched biological processes across all metamorphosis ancestors, suggesting functional convergence by independent evolution of distinct gene families involved in embryonic and post-embryonic development and nervous system differentiation. Evolutionary modelling further highlights a subset of these families exhibiting signatures of adaptive, lineage-specific gene family size increases associated with metamorphic development. These families include genes implicated in neural and sensory development, segmentation, and moulting. These findings support a model of the evolution of pancrustacean metamorphosis where distinct gene families from a common functional toolkit expand and are co-opted into facilitating transitions to multi-phasic life cycles. This reframes the role of moulting in arthropod diversification to be recognised as an important reservoir of genetic change that can potentiate truly remarkable life history transitions.

Social interactions are ubiquitous in nature and have the potential to affect trait evolution, particularly in group-living animals such as cooperative breeders. Interactions among conspecific individuals can affect the amount of additive genetic variation for a trait when the phenotype of an individual is also affected by the genotype of its social partner(s) via indirect genetic effects. Thus, quantifying both direct and indirect genetic effects of social partners is critical for understanding and predicting evolutionary trajectories. While much is known about maternal indirect genetic effects, empirical estimates of indirect genetic effects from other social partners remain limited, particularly in wild populations. Here, we use animal models to assess the contribution of indirect genetic effects from all social partners in a family group (mothers, fathers, and helpers) on juvenile morphometric traits across ontogeny in the cooperatively-breeding Florida scrub-jay (Aphelocoma coerulescens). We found indirect genetic effects of helpers and fathers on nestling weight, but no indirect genetic effect of mothers. Across ontogeny, we found increasing additive genetic variation in both weight and tarsus length. Our study provides a comprehensive assessment of within-group indirect genetic effects in a cooperative breeder and highlights the importance of considering indirect genetic effects beyond maternal effects.

The pea aphid (Acyrthosiphon pisum) is an important model organism for studying complex biological traits, including wing polyphenism and host-symbiont interactions, yet its regulatory genomic landscape remains largely uncharacterized. Here we present the first genome-wide chromatin accessibility map of the pea aphid, generated using the assay for transposase-accessible chromatin followed by sequencing (ATAC-seq). We profiled open chromatin regions (OCRs) in adult brains and late-stage embryos from winged and wingless morphs maintained under solitary or crowded conditions. We also paired ATAC-seq with RNA-seq in embryonic samples to examine the relationship between chromatin accessibility and gene expression. Libraries showed a high abundance of reads from the aphid endosymbionts Spiroplasma and Buchnera, reflecting preferential Tn5 transposase insertion into nucleosome-free bacterial DNA. After computational removal of these reads, the remaining aphid-mapping libraries displayed hallmarks of high-quality ATAC-seq data. We identified a consensus set of 37,127 OCRs enriched at promoters and distal regulatory elements, with substantial overlap with computationally predicted enhancers and enrichment for transcription factor binding motifs. Tissue identity was the dominant driver of chromatin variation, accounting for 85% of variance along the first principal component, with 19,513 differentially accessible regions distinguishing brain from embryo samples. By contrast, differences associated with wing morph or crowding treatment were modest. Promoter accessibility was significantly and positively correlated with gene expression genome-wide. Together, these data constitute a foundational regulatory genomics resource for the pea aphid and establish a framework for mechanistic studies of gene regulation in this ecologically and economically important insect.

Microplastics (MP) are widespread in aquatic ecosystems and pose a threat to freshwater biodiversity. While numerous studies examine physiological effects on aquatic organisms, less is known about how MP alter chemically mediated interactions that regulate predator-prey dynamics. Predator-induced defenses in Daphnia depend on detecting kairomones and represent an important form of adaptive phenotypic plasticity. Whether MP interfere with these responses, and through which mechanisms, remains unclear. Here, we show that polystyrene MP impair predator-induced defenses across Daphnia species by disrupting predator-cue-mediated plasticity at the behavioral, morphological, and molecular levels. In D. longicephala, chronic exposure to PS fragments weakened Notonecta-induced morphological defenses, whereas additive-containing PS fragments nearly suppressed defense formation and reduced body size. Consistent with these phenotypic effects, proteomic analyses revealed alterations in pathways related to molting and chitin metabolism, linking MP exposure to impaired defense formation. In D. magna, PS particles attenuated fish kairomone-induced diel vertical migration, with stronger effects for larger particles, consistent with reduced effective availability or perception of predator cues. Natural limestone particles caused only minor effects, indicating particle-specific rather than general particle-driven responses. Our findings demonstrate that MP can disrupt adaptive predator-prey interactions with potential cascading consequences for freshwater food webs.

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.

Cities are characterized by elevated temperatures, increased pollution, and high-density human populations which often are accompanied by changes in available resources, like food. These shifts have the potential to drive phenotypic divergence in urban wildlife. Functional morphological traits, like body size, can mediate interactions between wildlife and habitat and are closely tied to life history and fitness. While examples of functional morphological variation associated with urbanization are increasing, variation in such traits as a response to urbanization remains unexplored for most taxa. Here, we investigated morphological divergence between urban and rural populations of house mice (Mus musculus domesticus). House mice are globally distributed in diverse habitats and are a model system with a wealth of phenotypic data, making them useful for the study of the impacts of urbanization on morphology. Using a paired replicate design, we sampled urban and rural populations in three distinct metropolitan regions in the eastern United States. We found that body size was smaller in urban populations. Using 3D geometric morphometrics, we also analyzed variation in cranial shape across habitats. Differences in cranial shape were largely allometric, that is, driven by differences in body size. However, we also uncovered evidence of cranial shape variation between habitats not explained by size. In contrast, we did not find evidence for habitat-driven differences in cranial capacity independent of size. Overall, our results suggest a key role for body size in mediating morphological responses to urbanization and highlight the potential of house mice as a globally-distributed model for urbanization.

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.

The morphogenesis of complex vertebrate appendages requires precise regulation of growth, governed by distinct positional identities. The zebrafish caudal fin achieves a symmetrical, forked morphology through the regional specialization of the bony rays: peripheral rays are composed of relatively long, thick segments; while the central rays are made up of shorter, thinner segments, and their overall length is restricted. This length differential establishes the definitive forked shape of the organ. We asked whether these regional morphological differences reflect distinct underlying positional identities. Transcriptomic profiling of intact tissues from adult wild-type zebrafish suggested that central rays possess unique expression profiles, distinct from those of peripheral rays. We previously identified a treatment during embryogenesis that allows excess growth in the central rays, creating a truncate fin shape in adults-we asked whether this novel fin shape was caused by a peripheralization of the central rays. Indeed, the central rays of truncate fins were not only longer, but were composed of longer and thicker individual segments, reminiscent of peripheral rays. Further, gene expression in the central regions of truncate backgrounds showed signatures of peripheral identity. During development of the truncate phenotype, peripheral markers became expressed in more central domains of the growing truncate caudal fin, and in the supportive endoskeleton, the central hypural diastema was lost from the earliest stages. Ultimately, our results demonstrate how adult morphologies may be altered by shifts in positional identities. These findings clarify the anatomical patterning and molecular profiles that underlie regional specialization during caudal fin development.