Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

Hard cornification is the developmental process that produces the hardened epidermal structures that characterize tetrapod diversity, including scales, feathers, hair, and claws. This process relies on keratin proteins and additional reinforcing proteins that assemble, cross-link, and strengthen tissues to become mechanically resilient. Although the molecular composition of hard cornified tissues is known to vary among tetrapods, the program is least understood in amphibians. Within frogs, hard cornification occurs in the mouthparts of most tadpoles and in the nuptial pads, hindfoot claws, and foot tubercles of some adults. We characterized transcriptomic profiles of Bombina orientalis tadpoles at three developmental stages before, during, and after mouthpart cornification. We identified 25 keratin genes orthologous to amniote hard keratins in the B. bombina genome, 18 of which are upregulated in cornifying larval mouthparts. Furthermore, two of these hard keratin paralogs and an amphibian epidermal differentiation gene exhibit distinct spatial expression patterns within the jaw sheaths and keratodonts evidenced by in situ hybridization, suggesting they have discrete functional roles. The newly discovered diversity of the hard keratin repertoire in Bombina , and the concurrent expression of many of these paralogs in tadpole mouthparts, suggests that anuran hard cornification is much more complex than adult cornified traits thus far characterized in Xenopus frogs. Our results also challenge the notion that the diversification of keratins in Amphibia is primarily associated with the transition to more terrestrial habits, and may instead be shaped, in part, by the novel feeding apparatus of aquatic tadpoles.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.

Sperm competition can occur when sperm from two or more males compete for fertilization of a batch of eggs. While it has been extensively studied in internal fertilizers, it remains poorly under-stood in external fertilizers. To investigate how sperm competition affects fertilization under sperm limitation, we conducted controlled experiments on two sea urchin species: Paracentrotus lividus and Dendraster excentricus. Eggs from a single female were exposed to sperm from three individ-ual males separately (non-competitive treatment) or to a pooled sample from three males (competi-tive treatment). We measured fertilization success as proportion of eggs fertilized after two hours and assessed sperm motility as percentage of motile sperm and curvilinear velocity. For P. lividus , we estimated the sperm concentrations where fertilization success was 50% of maximum observed fertilization success (F50), and the fertilization efficiency of the sperm (Fe), i.e. the likelihood that a sperm - egg encounter results in a fertilizing contact, using the fertilization kinetics model of Styan (1998). For D. excentricus, fertilization success was measured at a sperm concentration expected to produce 50% fertilization success. Sperm competition reduced fertilization success and increased F50 compared to non-competitive treatments, suggesting antagonistic effects that may compromise fertilization under sperm-limited conditions in external fertilizers.

Biological populations have been evolving in multi-dimensional trait spaces. Ecological interactions can induce the evolutionary diversification of a biological population into distinct multiple populations, which is called evolutionary branching in adaptive dynamics theory. If possible mutations are restricted to certain directions due to developmental, physiological, or physical constraints, the resulting evolutionary dynamics may be restricted to subspaces with fewer dimensions than the original trait spaces. In general, such constraints are also evolving slowly, due to rare and slight mutational deviations from the constraint subspaces. In this sense, constraints are not strict. However, the method for describing non-strict constraints that is globally consistent has yet to be developed. This study proposes a general and flexible method for constructing a non-strict constraint subspace from the corresponding strict constraint subspace. This method enables efficient analysis of adaptive evolution including evolutionary branching under non-strict constraints in the framework of adaptive dynamics theory. Applying this method to a resource competition model shows that the shape of the carrying capacity distribution can be treated as a non-strict constraint curve, and that the likelihood of evolutionary branching is affected not only by the shape of the constraint curve but also by its slow evolution.

Food availability in natural habitats is often unpredictable and drives the selection of adaptive strategies in organisms to survive food scarcity. However, the genetic and molecular mechanisms that enable animals to survive under such nutritional extremes are poorly understood. Naturally adapted organisms therefore provide powerful genetic systems for uncovering the mechanisms that maintain energy homeostasis under nutritional extremes. Here, we leveraged the cavefish Astyanax mexicanus to identify a naturally occurring Thr166Met missense mutation in the melanocortin 3 receptor (mc3r) that reprograms energy homeostasis to facilitate adaptation to nutrient deprivation. To determine its functional consequences, we introduced the mutation into zebrafish mc3r using CRISPR/Cas9 genome editing. We show that adult mc3r mutants exhibit increased body size, adiposity, and hyperglycemia relative to wild-type animals, recapitulating key metabolic features of Tinaja cavefish and identifying mc3r as the first causal gene behind cavefish hyperglycemia. Investigations for early developmental consequences of the mutation revealed accelerated yolk utilization in mutant zebrafish larvae and strikingly elevated metabolic rates relative to WT, thus, uncovering a previously unrecognized role for mc3r in regulating energy expenditure. Importantly, the phenotype was also observed in Tinaja cavefish revealing a novel cavefish phenotype and establishing causal role for mc3r in elevated metabolic rate phenotype of cavefish. Together, these findings reveal that the Thr166Met variant produces distinct physiological consequences across life stages, enhanced energy storage and hyperglycemia in adulthood despite elevated larval energy expenditure. We propose that increased larval metabolism may support the energetic demands of enhanced foraging, whereas increased adiposity and blood glucose later in life provide energy reserves during periods of nutrient scarcity. More broadly, our study demonstrates how a single naturally evolved coding variant can reshape energy homeostasis across development and contribute to adaptation to nutrient-poor environments.

Corazonin is implicated in regulating diverse behavioral and physiological responses in insects, yet its role in honey bees remains unclear. Work in social and non-social species suggests an ancestral function for corazonin as a metabolic stress signal and a derived function as a pro-foraging signal in social insects. To investigate this hypothesis, we examined corazonin signaling across worker roles and metabolic states and tested its regulatory relationships with pathways central to worker division of labor. Central corazonin levels (both transcript and peptide) were approximately threefold higher in foragers than in nurses. Acute metabolic challenge did not alter brain corazonin but strongly upregulated fat body corazonin receptor (CrzR) transcript in nurses only, suggesting role-specific induction of peripheral sensitivity to corazonin signaling. Injection of synthetic corazonin decreased fat body insulin receptor substrate transcript, while transcripts of vitellogenin (Vg), unpaired-like A, ecdysone receptor B, and Cyp314a1 were unaffected. Measures of gustatory responsiveness, starvation resistance, and hemolymph sugar levels were similarly unaffected by corazonin injection. Reducing Vg through RNA interference in nurse-age bees did not affect CrzR transcript levels, despite vitellogenin's established roles in stress buffering, antioxidant defense, and lifespan. This contrast highlights the specificity of CrzR upregulation in nurses as a response to metabolic challenge rather than general stress. Together, these findings support corazonin as a co-opted nutritional signal that helps shape the forager physiological state, suggesting that it may act in part through interactions with insulin signaling rather than directly regulating classical nutritional or stress-response pathways.

Parasites decrease host fitness, more so during physiologically challenging life stages such as migration. However, parasites are unevenly distributed among individuals, and hosts with different physiological condition may face different costs of migration with infections. We tested whether the relationship between infection by haemosporidian parasites and oxidative balance during autumn migration in blackcaps depends on individual age and sex, two major sources of variation in prevalence and physiological condition. We captured blackcaps at stopover in central Spain and measured infection status and physiological parameters. Blackcaps physiologically more prepared to depart from stopover had lower antioxidant capacity of plasma, probably because of physiological adjustments prior to the upcoming flight stage. Plasmodium parasites occurred only in coinfections, which were more frequent among young blackcaps. Regardless of individual sex and age, coinfected birds had lower antioxidant capacity of plasma, and blackcaps coinfected with Plasmodium had more oxidative damage to lipids in their red blood cells. Our results support the idea that haemosporidian infections impair the oxidative balance of blackcaps during autumn migration, which could impair individual performance and ultimately fitness, with increased population impacts during the first year of life when a larger proportion of individuals migrate coinfected with harmful Plasmodium parasites.

A longstanding question in evolution is if similar morphologies shared by distantly related organisms facing similar environmental pressures belie similar functions. The carnivorous pitcher form has evolved in three distinct plant lineages and encompasses a suite of convergent morphological traits, providing a natural experiment to test hypothesized links between morphology and function. Multiple pitcher plant species possess fenestrations (i.e., small windows) that let light pass through the pitcher leaf. While research on one species, Nepenthes aristolochioides, demonstrates that fenestrations manipulate light to create false exits and increase prey capture, whether this convergent morphology serves this convergent function in other pitcher species remains unresolved. We explored the function of fenestrations in two additional species of pitcher plants, Nepenthes klossii and Cephalotus follicularis, where the morphology and function of fenestration were previously unexplored. We inspected micromorphology of pitcher structure, conducted an insect bioassay with live plants to test for links between morphology and function, and quantified traits associated with light manipulation from herbarium specimens. Both species exhibited fenestration and related pitcher morphology similar to N. aristolochioides. Yet our experiments did not reveal a significant effect of light passing through fenestrations on fly capture for these two species. Key differences between these species and the previously investigated species may be the presence of wax crystals and the absence of viscoelastic fluid. While future studies may uncover weak to moderate light manipulation function not detected in our study, our data do not support convergent function in these distantly related, morphologically similar species.

Early embryos must mount stress defenses without compromising the developmental programs that drive morphogenesis, but whether genetic background alters this balance remains unclear. We compared cellularizing Drosophila melanogaster embryos from inbred and outcrossed backgrounds under control conditions and after acute or chronic heat-stress. Both backgrounds developed successfully and showed modest transcriptomic differences at 18{degrees}C. Heat stress at 32{degrees}C, however, sharply separated their phenotypic and transcriptional responses. Inbred embryos showed poor larval hatching after acute and chronic heat, and this deficit persisted through adult eclosion. Their transcriptomes exhibited a greater stress-response burden, with enrichment of proteostasis, DNA repair, mitochondrial maintenance, RNA processing, and metabolic remodeling programs. Outcrossed embryos retained substantially greater developmental survival and higher abundance of transcripts associated with development, adhesion, epithelial organization, signaling, transport, sensory responses, and morphogenesis. Background-dependent divergence was greatest during acute heat. Under chronic heat, background-dependent differences were fewer, with most overlapping those seen during acute heat and a smaller set detected only under chronic heat. Transcript-origin analysis further showed that acute outcrossed-biased genes were predominantly maternally derived and showed no enrichment for purely zygotic transcripts, whereas chronic outcrossed-biased genes contained a larger zygotic component. In summary, the less viable inbred background mounted the broader molecular stress signature, whereas the more viable outcrossed background retained greater abundance of development-associated transcripts. These findings identify genetic background as a major determinant of how embryonic heat responses are distributed between molecular stress defense and developmental gene expression.

Fluctuating asymmetry (FA) is widely used as a proxy for developmental instability, although reduced rather than increased FA, has occasionally been reported under particularly challenging conditions, possibly because environmental filtering removes less developmentally stable individuals. Here, we investigated temporal variation in wing shape asymmetry in males of Drosophila sturtevanti collected in 2022, 2024, and 2026 at the same sampling site, an urban Atlantic Forest fragment. Sampling in 2022 and 2026 were completed during single field expeditions in early April, whereas in 2024 repeated collections were required until mid-May because of markedly low capture success. We therefore examined whether this difference was associated with unusual environmental conditions and whether FA was higher in 2024, as expected under greater developmental perturbation. Daily temperature and relative humidity were examined from early March onward, encompassing approximately one month before adult emergence. The 2024 sampling period was warmer and drier than the corresponding periods in 2022 and 2026 and coincided with the final phase of the strong 2023-2024 El Ni&ntildeo event. Contrary to our prediction, FA was significantly lower in 2024. The magnitude of directional asymmetry remained similar among years, although the spatial pattern of the DA vector differed between 2024 and 2026. The coincidence of reduced capture success and reduced FA is consistent with the hypothesis that adverse conditions may impose demographic filtering, such that individuals expressing greater developmental instability are less likely to contribute to the sampled adult population.

The genus Phakellia comprises predominantly fan-shaped deep-sea sponges, but its species diversity and boundaries remain unresolved. Here, we use an integrative approach combining morphological and molecular data to investigate the Phakellia robusta species complex and describe two new species, Phakellia pallida sp. nov. and Phakellia pusilla sp. nov. Overlap in external and spicular morphology makes these taxa challenging to identify. Molecular analyses based on mitochondrial (cox1) and nuclear (28S) markers support the recognition of the three taxa as distinct species, and reveal their close evolutionary relationship. The two new species co-occur in the NE Atlantic Ocean and the Mediterranean Sea, with overlapping depth ranges, although P. pallida sp. nov. is generally found at greater depths. Our results also suggest that several previous records of P. robusta from the Mediterranean and southern parts of the NE Atlantic may instead correspond to either P. pallida sp. nov. or P. pusilla sp. nov., indicating that its distribution requires reassessment. In addition, a group I intron previously reported in the cox1 gene of P. robusta was polymorphically detected at the same insertion site in some, but not all, individuals of P. pusilla sp. nov., but it was absent from P. pallida sp. nov. individuals. Overall, our results reveal pseudocryptic diversity within Phakellia and highlight the value of integrative approaches for resolving species boundaries and reassessing deep-sea sponge diversity and distributions.

Paraspinal muscles support and move the spine, and reduced size and density of these muscles is associated with increased risk of vertebral fracture and back pain. We test the hypothesis that subsistence-oriented forager-horticulturalists (Bolivian Tsimane) have larger and denser paraspinal muscles, and smaller age-related differences in paraspinal muscle size and density, than more sedentary post-industrialized suburbanites (Framingham Heart Study [FHS]). We measured cross-sectional area (CSA) and density of trunk extensors and psoas major at L3 from computed tomography scans of 411 FHS and 330 Tsimane (50% women, aged 50-90 years). Sex-stratified linear regressions determined associations of muscle CSA and density with age and cohort (Tsimane or FHS [ref.]), with and without an age-cohort interaction term. Covariates were height and weight. After adjusting for covariates, Tsimane women had 11-14% larger and denser extensors, and 17-26% smaller and less-dense psoas major, than FHS women. Tsimane men had 8-21% smaller and less-dense psoas major than FHS men. The age-related difference in extensors density was 41% smaller for Tsimane men than for FHS men. There were no other significant age-related cohort differences in muscle CSA or density. We observed a different balance in the size and density of trunk extensors versus psoas major between Tsimane and FHS. A possible explanation for this observation is cohort differences in the amount and types of physical activity, although future work is required to determine the influence of lifestyle and environmental factors on paraspinal muscle size and density.

The most studied symbiosis between land vertebrates and algae, is the one between Ambystoma maculatum and Oophila amblystomatis, where the latter colonizes the amphibian''s eggs. Although clear benefit given by algal growth for the salamander embryo has been observed, mutualism is not widely accepted. Mutualistic hypotheses posit benefits from the host to the algae which include protection, N- and C-sources, but no experimental evidence has been obtained. In fact, recent studies suggest that the algae are stressed during this relationship and propose fermentation rather than photosynthesis as main driver for algal growth. In this work, we analyzed embryonic host developmental rate, algal and embryo growth and photosynthetic function of eggs housed at different light intensities. We provide evidence that photosynthetic growth is the main driver for Oophila and that higher algal growth yields faster developmental rate and embryo size. Also, we provide strong evidence that rates of photosynthetic electron transport (and the concomitant O2 evolution) must be higher than those of consumption by the embryo for this symbiosis to be mutualistic. Since photosynthetic rate is dependent on light, this work illustrates the importance of light intensity in this symbiotic relationship.

Two rounds of whole-genome duplication (2R-WGD) in the vertebrate lineage are thought to have driven gene diversification. Mammals have four genes encoding five nebulin superfamily proteins (LASP1, LASP2/NEBL, NEB, and NRAP) hypothesised to have arisen from invertebrate LASP via WGD. Here, we examined the evolutionary fate of the nebulin superfamily after an additional WGD, teleost-specific genome duplication (TGD). Genome and transcriptome analysis of zebrafish (Danio rerio) and medaka (Oryzias latipes), of which the complete genomes were recently determined, showed only lasp family genes retained duplicate copies. Zebrafish retained two LASP2/NEBL-derived ohnologs; one (LASP2L) had been misidentified as a nebulette gene in the Zebrafish Information Network (ZFIN) despite lacking nebulette-specific exons. Another (LASP2/NEBL) is newly identified in this study because it lies in a newly determined region of the latest genome assembly. Contrastingly, medaka retained two LASP1 ohnologs and one LASP2/NEBL. The duplicated lasp family proteins showed overlapping and specific tissue expression patterns. We confirmed all three zebrafish lasp proteins bound actin and co-localized with actin-rich structures when ectopically expressed in cultured fibroblasts. This supports that TGD-derived lasp duplicates underwent lineage-specific retention and expression divergence in teleosts whilst conserving core actin-binding properties.

Coastal organisms experience natural and anthropogenic stressors simultaneously, yet the interactive effects of multiple stressors are largely unknown. In urbanized estuaries, coastal acidification (CA) is coupled to sewage effluent (SE): nutrient loading from SE stimulates microbial CO2 production, and this lowers the pH. The genomic targets of selection of CA and SE have been characterized separately but never together in early life stages of marine organisms. Here, we exposed eastern oyster (Crassostrea virginica) larvae to control, CA (~2800 atm pCO2), SE (5% by volume), and the combination (CASE) for 24 h across independent spawns, and used expressed exome capture sequencing to measure allele-frequency change before and after exposure, removing loci that also shifted in controls. CASE generally produced the lowest survival, although mortality was driven primarily by effluent and did not exceed the additive expectation. The size of allele-frequency change under CASE stayed close to the additive sum of the single stressors, but it showed compositional synergy: CASE had more outlier loci than CA and SE combined with 1,941 loci (747 genes) detected only under the combined treatment. CASE outliers were enriched for proteostasis, ciliary function, mitochondrial calcium handling, and a steroid-metabolizing (CYP17) module consistent with effluent endocrine disruptors. Selection acted repeatedly on standing variation across spawns and genetic backgrounds, with the largest allele-frequency shifts at rare, spawn-private loci. Enriched biological functions recurred across spawns more often than expected, while most outlier loci were specific to one or two spawns, consistent with genotypic redundancy. Our results show that combined stressors can recruit a compositionally synergistic genomic response, at loci and functions not detected under either stressor alone, even when survival and the size of allele-frequency change are additive.

Unlike most other heterotrophic protist lineages which synthesize glycogen, alveolates accumulate -glucan storage polysaccharides in the form of starch. Within Alveolata, ciliates do so by using a simple pathway analogous to that used for glycogen metabolism in eukaryotes. However, Myzozoa have evolved five additional enzymatic steps which are common to Dinoflagellata, Chromerids and Apicomplexa. Moreover, dinoflagellates have acquired seven additional enzymes that distinguish them from the Chromerids-Apicompexa lineages, while chromerids possess only a single enzyme not found in dinoflagellates. Such modifications are not found in other eukaryotic lineages save for those resulting directly from primary or secondary plastid endosymbiosis. The many differences affecting the starch pathway of Myzozoa can be explained by a single secondary or tertiary plastid endosymbiosis of a starch-containing alga followed by distinct compartmentalisation outcomes of starch metabolism in Dinoflagellata relative to chromerids and Apicomplexa.

Coleoid cephalopods (cuttlefish, octopus, and squid) are marine mollusks with elaborate nervous systems that support a diverse repertoire of complex behaviors. These include the neural control of the color, pattern, and texture of the skin, facilitating both adaptive camouflage and innate patterning that may reflect internal state. The development of transgenic cephalopods expressing fluorescent proteins, optogenetic actuators, and reporters of neural activity would contribute a new and important technology to cephalopod biology. The generation of transgenic cephalopods, however, has remained a major challenge. Here, we report the development of stable transgenic dwarf cuttlefish (Ascarosepion bandense) expressing ubiquitous nuclear-localized mScarlet, a red fluorescent protein. We evaluated multiple strategies for transgenesis, and established cuttlefish lines using both CRISPR and the transposons Sleeping Beauty and Minos. The stable expression of transgenes enabled live imaging of cell dynamics during embryonic development. The Minos transposon emerged as the most efficient transgenesis strategy and is adaptable to promoters and transgenes of choice. These strategies now enable the generation of diverse genetic tools for mechanistic studies of cephalopod biology.

While the influence of microorganisms in the thermal physiology of animals is increasingly recognised, the role of the gut microbiome in enhancing phenotypic plasticity remains underexplored, particularly in ectotherms. Here, we tested the effect of gut microbiomes in modulating the physiological response of lizards to sudden temperature fluctuations. In captivity, we conducted an experiment with wild-caught Podarcis lizards: Mediterranean Podarcis liolepis (n=9) adapted to warm and dry environments, and Atlantic P. muralis (n=18) adapted to cooler and wetter conditions. Animals were assigned to three groups (intervention, control-cold and control-warm) and subjected to microbiota manipulations using antibiotics and faecal microbiota transplants (FMT). In the intervention group, cold-adapted P. muralis received faecal suspension from warm-adapted P. liolepis, while control-cold and control-warm groups received intraspecific transplants. Microbiome variation was analysed through genome-resolved metagenomics of 162 faecal samples, yielding 313 MAGs across 15 phyla, containing almost 1M annotated genes. One week after transplantation, microbiomes of control animals differed from their original state, whereas those of the intervention group (P. muralis), transitioned to a state resembling P. liolepis. After two weeks, both control groups showed trends of reversing their microbiomes to their original states. However, the intervention group displayed a distinct trajectory, resembling the microbial donors'. Although potentially relevant trends were observed, these microbiome alterations did not translate into detectable significant differences in their thermal physiology. Nevertheless, our results provide proof of concept for microbial transplants in lizards, and opportunities to further explore the link between gut microbes and host physiological responses in ectotherms.

Animal behaviour is both innately constrained and shaped by learning. This mosaic organization has evolved in response to species-specific ecological demands and may differ between sensory modalities. Flower-visiting animals are a particularly useful system for investigating the relationship between sensory ecology and learning because they rely on multiple floral cues, particularly odour and colour, to locate food sources. However, it remains largely unexplored whether specialization on floral resources entails divergence in learning abilities across sensory modalities. Drosophila elegans is a flower-feeding species that depends heavily on floral resources throughout its life; adults spend much of their time on flowers and larvae develop on fallen flower leaves. Here, we compared odour-reward and colour-reward associative learning between the flower-feeding D. elegans and the fruit-feeding D. melanogaster. We found that, under conditions of equilibrated motivation, odour- and colour-preference, and using the same sugar reward, D. elegans exhibited poorer odour-reward learning performance but better colour-reward learning performance than D. melanogaster. These results suggest that the modality-specific eligibility of sensory information to enter into associations, known as the 'Garcia-effect' in experimental psychology, can evolve oppositely between species. This highlights the relationship between ecological specialization and mnemonic processing, and shows that biological 'intelligence' is not general.

Primordial germ cells (PGCs) are the stem cell lineage responsible for transmitting genetic information across generations and are therefore central to studies of germline development, reproduction, and evolution. However, a lack of molecular tools in jawless vertebrates such as lampreys and hagfish has limited comparative insights into the evolutionary origins of vertebrate germline cells. Here, we report the development of the first germline-specific reporter (GSR) in the sea lamprey (Petromyzon marinus), Tg(piwil1: egfp-UTRnanos1), a piwil1-based germline reporter that is driven by the sea lamprey piwil1 regulatory region that directs Green Fluorescent Protein (GFP) expression in early PGCs. Promoter activity was validated by robust GFP expression in PGCs of zebrafish embryos co-injected with Tol2 transposase mRNA. Injection of the construct into sea lamprey embryos resulted in early, persistent, and gonad-localized GFP-positive cells. These results demonstrate stable germline-specific transgene expression in a jawless vertebrate and suggest deep evolutionary conservation of germline regulatory mechanisms between jawless and jawed vertebrates. Furthermore, the piwil1-based germline reporter line enabled lifetime visualization of zebrafish germline cells, revealing that increased PGC abundance during embryogenesis biases toward female sexual differentiation. Together, this study establishes the first GSR system in a jawless vertebrate and provides a useful platform for investigating germline biology and vertebrate germline evolution.