Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

High and low tides occur twice a day (every [~]12.4 hours), with the largest tidal ranges during spring tides around new and full moons (every [~]14.765 days). While these lunar cycles are known to influence many animal phenotypes, particularly the reproduction of coastal animals, the genetic basis of lunar-related rhythms remains unclear. Since phenotypic variation is a valuable resource for elucidating such mechanisms, we examined geographic variation in the lunar-regulated mass spawning of the grass puffer (Takifugu alboplumbeus) along the Japanese coast. We found that western populations spawn during the first half of the spring tides, whereas eastern populations spawn during the second half. Furthermore, although spawning typically occurs a few hours before high tide, this timing is restricted to a specific time window that is earlier in the western populations than in the eastern ones. Behavioral analysis of larvae also revealed a shorter free-running circadian period ({tau}) in the western population than in the eastern ones. As differences in {tau} affect individual variation in the timing of physiological functions and behaviors, we hypothesized that differences in {tau} could account for the different time windows and consequently the observed difference in spawning days. Population genomics analysis identified proline-rich transmembrane protein 1-like (prrt1l) as a candidate gene. Expression of prrt1l was observed in the circadian pacemaker suprachiasmatic nucleus, and triple CRISPR F0 knockout of prrt1l shortened the free-running period in larvae. These findings suggest a potential mechanism underlying the geographic variation in lunar-synchronized spawning behavior. HighlightsO_LIThe geographic variation exists in the lunar-regulated spawning of the grass puffer, with differences in spawning dates and times between western and eastern Japan. C_LIO_LIThe free-running period of western populations is shorter than that of eastern populations, which is consistent with their earlier spawning timing. C_LIO_LIPopulation genomics analysis identified prrt1l as a candidate gene harboring population-specific missense mutations, the knockout of which shortens the free-running period. C_LI

The evolution of body shape reflects the interplay between functional constraints and habitat structure. In fishes, cave environments are well known for promoting regressive traits such as eye and pigment loss, yet their influence on overall body form remains poorly understood. Here, we examine patterns of body shape variation in cave- and surface-dwelling trichomycterid catfishes from northeastern Colombia to assess whether consistent associations exist between habitat type and morphology. Using geometric morphometric analyses, we quantified differences in body shape among species inhabiting subterranean and surface environments. Our results reveal significant habitat-associated differentiation in body shape along the main axes of morphological variation. Cave-dwelling species exhibit more elongated and fusiform body shapes, whereas surface-dwelling species tend to show deeper and more robust morphologies. In a functional context, these contrasting body patterns suggest associations with differing locomotor demands imposed by subterranean versus surface habitats. Although we do not explicitly test convergence or performance, the recurrence of similar body shapes among species from different clades occupying comparable habitats is consistent with repeated morphological responses to shared ecological constraints. Research HighligthsO_LIMultivariate shape analyses reveal significant habitat-associated variation in trichomycterid fishes. Recurrent morphological patterns suggest repeated responses potentially mediated by habitat constraints. C_LIO_LIBody shape differs consistently between cave- and surface-dwelling trichomycterids. Cave species exhibit more elongated and fusiform forms, whereas surface species display deeper body configurations. C_LI

A major challenge to investigating the proximate causes of ecological adaptation is the difficulty of studying the phenotypes of organisms in their natural environments. By necessity, many studies seeking to determine the genetic and cellular basis of adaptation therefore investigate potentially adaptive phenotypes under laboratory conditions where organisms are more easily experimentally manipulated. For laboratory models, it remains unclear if organisms maintained long term under laboratory conditions are representative of relatives in their natural environment. In recent years, Drosophila sechellia, a specialist species endemic to the Seychelles, has emerged as a (neuro)genetic model for studying the molecular basis of ecological adaptation. A multitude of studies have investigated the genetic and cellular basis of various aspects of this species specialization in a laboratory setting. However, the vast majority of these studies use laboratory strains of D. sechellia that were collected many decades ago, and have been maintained under conditions very different from their natural niche. Thus, it remains unclear if and how these strains resemble their wild counterparts. Here, we compare the phenotypes of these laboratory strains with recently-collected wild D. sechellia to ask if laboratory strains display a loss or degradation of phenotypes potentially involved in their specialization resulting from their long-term laboratory maintenance. Across several behavioral and anatomical phenotypes, we find a high degree of similarity between wild-caught and laboratory-maintained strains. Our results suggest that studies of the molecular mechanisms underlying D. sechellias phenotypes associated with specialization are likely representative of the evolution of these flies in the wild.

Astyanax mexicanus consists of eyed, river-dwelling "surface" fish, and multiple, independently evolved cave populations, which have converged on troglobitic traits such as eye loss and reduced metabolism. However, considerably less is known about constructive adaptations, which include a larger olfactory epithelium in cavefish. It is unknown how this relates to the olfactory bulb (OB), which is the first stage of olfactory sensory processing in the brain. The goal of the present study is to begin to define the structure and functional organization of the OB in A. mexicanus, and to begin to understand how it was transformed via cave adaptation. We addressed these questions using whole-mount immunohistochemistry and in vivo Ca2+ imaging from the OB of developmentally matched surface and Pachon cavefish. The cavefish OB was significantly larger than surface fish by 14 days post fertilization (dpf), which was accompanied by a broad and proportional increase in synaptic input to most glomerular regions. Increases in the size of the OB were accompanied by increases in the number of neurons expressing tyrosine hydroxylase and calretinin, the latter of which occurred primarily in the medial OB and could not be explained as a compensatory response to a larger OB. In vivo Ca2+ imaging from the dorsal OB of surface and cavefish in response to a panel of chemical stimuli revealed odor-evoked responses that were spatially organized and highly conserved across the two populations. Surprisingly, the medial OB was consistently activated by any change in water flow in both populations, although the number of water-responsive neurons was significantly greater in cavefish when measurements were performed using either in vivo imaging or the neuronal activity marker phospho-ERK. Water-responding neurons were similarly present in the olfactory epithelium in both populations, along with neurons expressing the mechanosensitive ion channel Piezo2, with significantly more Piezo2-expressing neurons present in cavefish. Therefore, cavefish exhibit enhanced multisensory integration of olfactory and mechanosensory input in the earliest stage of olfactory sensory processing in the brain.

The olfactory system plays a critical role in mammalian environmental perception, with some clades relying on an expanded accessory olfactory (vomeronasal) system (VNS) to detect larger, non-volatile odorants. Mammals make extensive use of this system for social communication between conspecifics. Recent studies have begun to investigate how the VNS changes in response to or as part of ecological transitions. Several studies have identified trends of VNS-associated gene loss or regression in secondarily aquatic mammals. However, continuing discussion on genotype-phenotype correlation within the VNS means that greater effort should be made to investigate the morphology of the VNS in species where it remains poorly understood. Here, we use skeletal and soft-tissue data to demonstrate that the vomeronasal groove, an established osteological correlate for the VNO in bats and primates, is also a valid indicator for its presence in Caniformia. Additionally, we confirm the presence of the VNO in the secondarily aquatic North American river otter (Lontra canadensis) and compare its morphology with that of two close-related species, the semi-aquatic American mink (Neogale vison) and the terrestrial long-tailed weasel (Neogale frenata). This study expands the valid taxonomic scope of the vomeronasal grooves proxy as an osteological correlate, confirms the presence of the VNO in the previously undescribed system of the North American river otter, and highlights the complexity of the mammalian accessory olfactory system.

Dental enamel, the hardest mineralised tissue in the human body, has proven to be an excellent source of ancient proteins, which have been found to survive within dental enamel for at least twenty million years. In archaeological and palaeontological contexts, the enamel proteome is generally considered to be rather small, consisting of about twelve proteins, most of which are unique to enamel. During amelogenesis these proteins undergo in vivo digestion by matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) as well as serine phosphorylation by family with sequence similarity member 20-C (FAM20C) that alter their characteristics. Gaining knowledge of the previously understudied influence of amelogenesis on the archaeological human dental enamel proteome could benefit various palaeoproteomic analysis, especially in an human evolutionary context. Here we present archaeological dental enamel proteomes and explore protein cleavage patterns and sequence coverage to estimate the effects of in vivo digestion, as well as explore phosphorylation patterns. Additionally, we present a new marker based on phosphorylation to estimate genetic sex.

Echinoderms possess a complex immune system, primarily relying on coelomocytes - immune cells circulating in coelomic fluids. Over the last few decades, various coelomocytes have been described based on morphological features, with holothuroids exhibiting the highest diversity of cell morphotypes among the different echinoderm classes. However, while the overall immune function of these cells is broadly accepted, their respective functions remain unclear, and molecular data specific to the different cell types are still limited in the literature. In this study, we address this gap in functional information and molecular data by using single-cell RNA sequencing (scRNA-seq) on coelomocytes from the perivisceral fluid of Holothuria forskali. We identified 10 distinct clusters, each assumed to correspond to a distinct transcriptional coelomocyte population. Among these, cluster 0 occupies a central position relative to the others, suggesting it may represent "progenitor cells", whereas cluster 6 is markedly divergent from all other clusters. Functional enrichment analyses revealed that some clusters ensure key immune functions, including pathogen recognition, phagocytosis, complement activation and redox balance regulation. In addition, examination of the processed samples under a microscope confirms the presence of a small proportion of recently discovered carotenocytes (7.0%) in the perivisceral fluid, a cell type rich in carotenoids. By using transcriptomics data previously obtained for this cell type by bulk RNA sequencing (bRNA-seq), it was possible to confidently identify cluster 6 as carotenocytes and provide further insights into their gene expression. While further analyses are needed to link other clusters to the different morphotypes previously described in the literature, this pioneer study presents preliminary data on the functional diversity of holothuroid coelomocytes, which could be of broad interest for a better understanding of holothuroid immunity as well as for the study of immune cell lineage evolution across deuterostomes.

We examined how thermal shifts influence development time and adult body size in Drosophila melanogaster. Individual flies were exposed to alternating temperatures of 25{degrees}C (optimal) and 17{degrees}C (cold), with shifts introduced at key developmental transitions: larval hatching and pupariation. We found while larval-stage temperature is the biggest determinant of thermal plasticity of development time and adult size, the egg-stage temperature also influences the pace of development and growth throughout pre-adult duration. The effect of low-to-high and high-to-low temperature shifts on development and growth may not be symmetric. When eggs are reared at 25{degrees}C and then shifted to 17{degrees}C, larval and pupal durations undergo reduction compared to constant 17{degrees}C, but it produces slightly larger adults. A higher egg-stage temperature thus seem to exert a carryover effect that accelerates subsequent development and growth when later stages experience colder temperatures. Surprisingly, flies whose egg stage is exposed to 17{degrees}C followed by a shift to 25{degrees}C also have reduced larval duration and larger size, relative to those developing at constant 25{degrees}C. We speculate this could be either because 17{degrees}C to 25{degrees}C represents a low-to-high temperature shift or a sub-optimal-to-optimal thermal shift that results in metabolic and/or hormonal changes accelerating differentiation and growth. While pupal duration is sensitive to current and to some extent prior thermal environments, it does not contribute substantially to thermal plasticity of size. Development time is longer in males than in females, and this difference seems to start from larval stage while the pupal duration plays a bigger role in creating this sex-specific difference. Overall, employing individual fly rearing, this study helped to unravel the effect of thermal shifts on growth and development in D. melanogaster with great precision.

Lysosome-rich enterocytes (LREs) are a specialized population of intestinal cells that mediate the uptake and absorption of dietary proteins in fish and neonatal mammals. Loss of LRE function causes inhibited growth and poor survival due to protein malnutrition. Previously, we reported that in zebrafish loss of Plasmolipin (pllp), an endosomal membrane protein highly expressed in LREs, impairs LRE differentiation and dietary protein absorption, resulting in marked reduction in survival rates. Raising pllp homozygous mutants surviving to adulthood and in-crossing them for multiple generations resulted in their adaptation to malnutrition, with adapted pllp mutants showing no survival deficits. To uncover mechanisms underlying this phenomenon, we compared the older adapted pllp allele with genetically related wild type (WT) fish and a newly generated pllp mutant allele. Using transcriptome profiling and quantitative protein absorption assays, we found that adapted pllp mutants exhibit upregulation of LRE endocytic components, resulting in a capacity for protein absorption that exceeds that of WT. This hyperactivation of LRE endocytic and absorptive activity is aided by a fine-tuned transcriptional regulation of immune genes that may contribute to the enhanced survival of pllp mutants in the face of increased exposure to environmental antigens. Genetic analyses indicate that these adaptations emerge gradually and are recurrent as shown experimentally by the adaptation of a mutant allele upon inbreeding and natural selection. Overall, our study illustrates that genome wide transcriptional changes underly adaptation mechanisms that enhance intestinal function and organismal survival in response to protein malnutrition. Author SummaryIn this study, we investigated how zebrafish adapt to protein malnutrition when the function of specialized intestinal protein-absorbing enterocytes, also found in newborn mammals, is impaired. We observed that fish carrying a mutation that severely disrupts intestinal protein absorption gradually recovered their ability to survive over multiple generations of inbreeding, even though the underlying mutation remained intact. By comparing gene activity in these adapted fish with that of newly generated mutants, we found that adaptation involves a coordinated rewiring of two systems: the enterocytes themselves became hyperactivated, absorbing more protein than even wildtype fish, while the immune system was simultaneously recalibrated to dampen inflammation. We further showed that this adaptive process is recurrent by using a second, independently generated mutant line that underwent a strikingly similar recovery trajectory over successive generations. Together, our findings reveal that animals can overcome a severe, heritable nutritional deficit through a gradual, genome-wide transcriptional response that fundamentally reshapes intestinal function across generations.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

Sarita Lake, British Columbia houses a distinctive population of threespine stickleback (Gastrosteus aculeatus L.) with a phenotype characterized by unusually large individuals relative to nearby conspecifics. We tested the hypothesis that members of this population are not isometrically larger but rather exhibit variation in allometric trajectories that reflect changes in developmental timing impacting the developmental-genetic architecture of the phenotype. We used 3D geometric morphometrics to characterize the size and shape of skulls, pectoral girdles and pelvic girdles from a sample of individuals from nearby freshwater and marine populations and compare them to a sample from Sarita Lake. We showed that individuals from the Sarita Lake population are larger in each body region compared to most other populations examined. Further, these individuals have dorsally expanded skulls and relatively robust pelvic armour. We also showed that the relationship between size and shape is differently structured among body regions and is heavily influenced by non-uniform sexually-mediated variation across populations sampled. Our results reflect complex underlying developmental trajectories, and we suggest that the large phenotype observed may be driven by fecundity selection on female size in combination with a limnetic trophic niche and relatively increased predation pressure in Sarita Lake.

Harbour porpoises (Phocoena phocoena) in the North and Baltic Seas are increasingly impacted by anthropogenic pressures, including underwater noise, fisheries and pollution. These pressures correlate with declining population health, particularly affecting the respiratory system. Growing pathological lesions, partly resulting from high prevalence of parasitic infestations and subsequent diseases, can impair tissue function and oxygen supply to distant end-organs. In this study, we applied an integrative MultiOmics approach (proteomics, metabolomics, lipidomics) to analyse the lungs and muscles of 12 wild harbour porpoises with compromised respiratory health. Our aim was to identify dysregulated biological pathways across omics layers to advance insights into adaptive physiological responses and to define disease-associated molecular signatures that could assist health assessments. Our analysis revealed pronounced immune system and antioxidative responses in the lungs and muscles, indicated by enhanced immunoglobulins, plasmalogens and glutathione-related proteins. In the lungs, high cardiolipin levels and reduced collagen suggest impaired tissue structure and function, while tissue maintenance processes were elevated in the muscle. Both tissues exhibited metabolic alterations suggestive of energetic imbalance, including increased purine metabolism in the lung and decreased lipid metabolism in the muscle. Several dysregulated molecules were shared across tissues, pointing to pathophysiological effects. The proposed disease-associated molecular signatures included the protein SLC25A4, the metabolite O-phosphoethanolamine and the lipid TG O-16:0_16:0_20:4 for the lung, and the protein SPEG, the metabolite pipecolic acid, and the lipid BMP 18:1_22:6 in the muscle. Our findings elucidate the complexity of molecular mechanisms linking anthropogenic and environmental stressors with vulnerability and resilience in a marine sentinel species. Furthermore, this study highlights the potential of integrative omics to define disease-related marker panels, thereby supporting ongoing and future health monitoring and conservation efforts.

Light and transmission electron microscopy were used to identify changes in ultrastructure of the olfactory pit of larval zebrafish (Danio rerio) that occur as a result of age and altered environmental light levels. Larvae were reared under control/cyclic light or constant light condition until 4, 8, and 15 days postfertilization (dpf). The larval olfactory pit consisted of an epithelium that varies from simple to pseudostratified to stratified and contained three types of receptor cells: ciliated, microvillar and ciliated crypt. A variety of non-receptor cells were also identified: kinociliate non-sensory supporting cells, vesicular supporting cells, basal cells and an occasional intruder, such as a neutrophil or a lymphocyte. Microvilli projecting from microvillus receptor, kinociliate, and vesicular supporting cells were single, forked, or doubly forked. Junctional complexes were evident between a variety of cells including adjacent epidermal cells, an epidermal cell and a kinociliate cell, a kinociliate and a vesicular supporting cell, and two vesicular supporting cells. Desmosomes were also observed between adjacent cell types. With age, the olfactory epithelium thinned and vesicle number varied. In larvae reared in constant light, mitotic figures were evident, microvillar receptor cells were absent, and, at 4 dpf, some ultrastructural components were similar to those observed in 8 dpf control animals, suggesting precocious development. These findings suggest that constant light rearing alters the timing of receptor replacement, supporting previous work showing that rearing light levels impact sensory system growth and development.

The cockroach Blattella germanica possesses panoistic ovaries, in which oocytes lack nurse cells and therefore need to rely on their own transcriptional activity to support embryogenesis. Ovarian development in this species involves the development of a single basal ovarian follicle (BOF) per gonadotropic cycle, a process strictly regulated by endocrine signals, primarily juvenile hormone and ecdysone, which act at both the transcriptional and translational levels. In addition, transcriptional activity in these ovaries is necessary for both regulating and genome protection, and at this level, PIWI-interacting RNAs (piRNAs) play an essential role. Although insect ovaries are known to be particularly rich in piRNAs, their function in ovary maturation is still not well defined. For this purpose, we characterize the piRNA expression dynamics across seven key developmental and reproductive stages, ranging from late nymphal instars to post-vitellogenic adults. piRNA expression in B. germanica shows coordinated fluctuations. Expression remains stable in previtellogenic ovaries, whereas vitellogenic ovaries show pronounced changes. Moreover, vitellogenic ovaries exhibit reduced piRNA diversity due to strong enrichment of a subset of highly expressed piRNAs. Our data show that although piRNAs predominantly map to transposable elements, particularly LINEs, there is a notable increase in gene-derived piRNAs toward the end of the cycle. Our results suggest regulatory roles of piRNAs in modulating both TEs and mRNAs during BOF maturation, likely related to changes in the follicular cell program.

Ancient human dental calculus is one of the richest archives of archaeological biomolecular information, providing direct evidence of diet, oral health, and the oral microbiome. Proteomic analyses of this biological matrix have so far focused mainly on oral microbes and dietary proteins, with milk proteins such as beta-lactoglobulin (BLG) providing the largest corpus of proteomic evidence. Despite the close relation between the various stages of dental calculus formation and mineralization with the dental enamel surface, proteins from the dental enamel matrix have not previously been reported outside of dental enamel tissue. Here we reanalysed 498 ancient dental calculus proteomes from 14 published studies (n=434 individuals) reporting the presence of BLG, spanning from the Neolithic to the Victorian Era and applying different protein extraction protocols (FASP, GASP, SP3 and in-solution digestion). Dental enamel matrix proteins were identified in ten studies (n=37 individuals), with amelogenin being the most frequently detected. Enamel peptides occurred more often in studies that applied SP3, although amelogenin was successfully identified through both SP3 and FASP. Structural proteins, including enamelin, ameloblastin, and MMP20, were also identified. The detection of AMELX and AMELY peptide sequences provided new insights into cases where the sex was previously undetermined. These findings establish dental enamel proteins as a new category of biomolecules detected in dental calculus, broadening its application beyond diet and microbiome studies to possible sex estimation. HighlightsO_LIDental calculus entraps oral microbes along with endogenous and exogenous particles during formation and mineralization C_LIO_LIWe conduct reanalysis of 14 published ancient dental calculus studies (n = 434 individuals) spanning the Neolithic to Victorian Era C_LIO_LIDental enamel proteins AMELX, AMELY, AMBN, COL17A1, ENAM and MMP20 are identified in ancient human dental calculus C_LIO_LIAmelogenin was the most frequently detected enamel protein C_LIO_LIWe expand dental calculus palaeoproteomics beyond diet and oral microbiome to potentially include sex estimation C_LI

Skeletal muscle plays important locomotive and metabolic functions, yet its formation and maintenance are processes remaining largely unclear mechanistically in any animal. Teleost fishes display extraordinary muscle growth due to their ability to undergo both hyperplasia and hypertrophy throughout life. These phenomena vary greatly even between closely related species, providing opportunities to elucidate growth dynamics and underlying mechanisms through cross-species comparisons. Using histological and genetic approaches, we compared muscle growth dynamics in three closely related danionin species with distinct growth capacities: the giant danio (Devario malabaricus), the zebrafish (Danio rerio), and Danionella cerebrum, as well as the more distantly related African turquoise killifish (Nothobranchius furzeri). Our study reveals alterations in spatial patterning of muscle hyperplasia and developmental timing to be major contributors to observed differences in muscle growth between examined species. Single-cell RNA profiling, in situ hybridization chain reaction and cell type-specific mutagenesis revealed muscle stem cell-specific expression of extracellular matrix genes that mediate stem cell activity, which in turn may drive growth differences between species. Taken together, our findings highlight autonomous regulation of muscle stem cells as a conserved but adaptable mechanism governing muscle patterning and diversification.

Arthropods must periodically moult their exoskeleton to permit growth, a conserved developmental process whose morphological and behavioural execution varies widely among lineages. Horseshoe crabs (Limulidae) are members of Xiphosura, a chelicerate lineage with a fossil record extending as far back as the Ordovician and provide a valuable comparative framework for studying the evolution of moulting strategies in Arthropoda. Despite their importance, detailed morphological characterization of moulting in horseshoe crabs remains scarce, limiting developmental studies and broader comparative analyses. Here, we provide a detailed morphological characterization of the moulting process in the Atlantic horseshoe crab Limulus polyphemus. Morphological changes in specific anatomical structures, including the anterior margin of the prosoma, lateral spines and dorsal spinous process of the opisthosoma, were observed during the moulting process. By tracking these morphological markers, such as retraction of the epidermis from the cuticle and degree of corrugation of the epidermis, we were able to identify individuals in the early and late pre-moult stage, predict the onset of ecdysis, and distinguish post-moult and intermoult stages. We compare ecdysis patterns in L. polyphemus with other arthropod taxa, both extant and fossil. We find that, despite differences in behavioural execution, ecdysis in L. polyphemus shares features with other chelicerates, and that both phylogenetic signal and convergent patterns are evident across Arthropoda. This study offers a robust, non-invasive method for determining moult stages in juvenile horseshoe crabs and provides insights into diversity and constraints of ecdysis in Arthropoda.

Garra rufa, commonly known as the doctor fish, is a small freshwater cyprinid notable for its exceptional tolerance to high temperatures, surviving even at around the human body temperature of 37 {degrees}C, and has emerging potential as a novel laboratory model for human cancer xenotransplantation and infectious disease research. To establish a foundation for its experimental use, we conducted comprehensive anatomical and histological analyses across major organ systems. The overall body organization and tissue architecture of G. rufa are broadly similar to those of zebrafish (Danio rerio), indicating a conserved cyprinid body plan. However, several organ systems in G. rufa exhibited species-specific differences compared with zebrafish, including a well-developed adhesive disc around the oral region, a long and coiled intestine, and a distinct dark pigmentation of the peritoneum. These species-specific traits may reflect ecological and behavioral adaptations of G. rufa, including benthic scraping in warm, flowing habitats. Physiological assays confirmed that G. rufa maintains high survival rates and normal swimming activity at 37 {degrees}C, whereas zebrafish exhibit significant mortality and reduced locomotion under the same conditions. Collectively, this work provides a comprehensive histo-anatomical atlas of G. rufa, highlighting its unique morphological specializations while establishing an essential reference for the development of this species as a novel experimental fish model.

Habitat transitions are a major driver of morphological evolution. Teleost fishes have repeatedly transitioned from benthic to pelagic habitats, often evolving predictable changes in body shape that enhance hydrodynamic efficiency. While freshwater sculpins (Cottidae, Perciformes) are usually benthic, two genera in Lake Baikal, Comephorus and Cottocomephorus, have independently evolved into midwater niches. As sculpins lack a swim bladder, these lineages instead improved buoyancy through reduced skeletal density and increased lipid stores. Using micro-computed tomography and two-dimensional morphometrics, we characterized skeletal evolution across the Baikal sculpin radiation. We found that parallel changes in bone mineral density and microstructure independently evolved in the two pelagic clades. Density reductions occurred throughout the skull in pelagic species. The basibranchials and neurocranium exhibited the lowest overall bone density across all cranial elements. While the jaws maintained the highest absolute density values among the bones we measured, they also showed the greatest proportional reduction in density associated with pelagic habitat use, with a 56.86% decrease in percentage hydroxyapatite and a 21.39% increase in porosity. Morphometric analyses further identified convergence toward an elongate body shape, reduced and posteriorly shifted eyes, and elevated fin insertion in pelagic taxa. These results demonstrate a repeated skeletal lightening and body shape changes accompanying benthic-to-pelagic transitions. This pattern mirrors other benthic-to-pelagic transitions in teleosts that lack swim bladders, highlighting shared biomechanical and microstructural solutions to life in the open water.

The Meckels cartilage (MC) is a fundamental component of mandibular development across vertebrates. In mammals, MC is transient and functions primarily as an early template for mandibular ossification, whereas other vertebrates, including zebrafish, retain MC within the mandible throughout life. Despite its importance, the requirements for MC in sustaining mandibular growth and how signaling pathways implicated in MC development contribute to this process remain unclear. Here, we investigated the dosage-dependent roles of BMP antagonists during zebrafish MC development using mutant alleles of grem1a, nog2, and nog3. Compound mutant adults exhibited fully penetrant mandibular truncation. MC shortening emerged after early larval stages, indicating a requirement for BMP antagonism to sustain cartilage growth. Chondrocyte number remained unchanged as phenotypes developed, but mutants displayed disorganized cartilage morphology and increased chondrocyte volume. Molecular analyses revealed reduced col2a1a domains and expanded ihha and col10a1a expression, consistent with ectopic hypertrophic-like differentiation. Constitutive activation of BMP receptor signaling in chondrocytes recapitulated these phenotypes. Although osteogenesis appeared unaffected by 14 dpf, loss of a tnn skeletal mesenchyme population was observed. Together, these findings demonstrate that BMP antagonists sustain MC growth by regulating chondrocyte differentiation and cartilage organization to support mandibular growth in non-mammalian vertebrates. Summary StatementThis study leverages zebrafish to define the cellular and molecular mechanisms by which BMP antagonism sustains mandibular growth.