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As a flagship taxa for marine ecosystems, seahorses possess a unique reproductive strategy of "male pregnancy". They are severely threatened by marine petroleum-based pollution but the molecular mechanism involved remains unclear. We evaluated the toxic effects and mechanisms of sub-acute exposure to benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon (PAH), at three environmental related dosages (0.5, 5, and 50 μg/L) on the reproductive organs of sex-role-reversed lined seahorses (Hippocampus erectus). BaP induced ovary, testis and brood pouch tissue damage in a concentration-dependent manner. Transcriptomic results suggested that reproductive organs of the seahorse could undergo biotransformation and detoxification of BaP via the P450 enzyme system; the differential expression of key genes related to these pathways determined the differences in the accumulation of toxic substances in the reproductive organs. Moreover, toxic substances directly induced differential tissue damage in situ by activating tissue-specific signaling pathways: BaP-induced ovarian apoptosis and failure by upregulation of the pro-apoptotic genes, vadc1, traf2b, tnfsf10, and pycard (P < 0.05); inhibition of testicular function through disruption of genes associated with "ECM-receptor interaction", "Tight junction", and "Spermatogenesis" pathways; and interference with brood pouch immune responses, significantly suppressing the expression of ripk1, il-1b, casp3a, apaf1, calr, and canx (P < 0.05), thereby impairing "Apoptosis", "Phagosome", and "Necroptosis" processes, ultimately compromising brood pouch maintenance. Toxic substances exacerbate damage to the reproductive organs in seahorses by disrupting Ca 2+ homeostasis. At environmentally-relevant concentrations of BaP, the reproductive efficiency of seahorses may be severely affected, increasing the risk of a decline in the abundance and diversity of wild populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The great seahorse ( Hippocampus kelloggi ) is one of the larger species within the seahorse group and is widely distributed in coastal areas of the Indo-Pacific. However, the natural resources of this species continue to decrease, rendering it a vulnerable species that faces a high risk of extinction. Therefore, there is an urgent need to conduct research on the genetic diversity of this species to protect its genetic resources. In this study, we conducted whole-genome re-sequencing (WGRS) on three H. kelloggi populations from the Red Sea (RS, n = 30), the Andaman Sea (AS, n = 13), and the South China Sea (SCS, n = 13), and a total of 1,398,936 high-quality single-nucleotide polymorphisms (SNPs) were identified. The results indicate that the average observed heterozygosity ( Ho ) and the average expected heterozygosity ( He ) for the RS, AS, and SCS populations are 0.2031 and 0.1987, 0.1914 and 0.1822, and 0.2083 and 0.2001, respectively. The three geographic populations exhibit a high degree of genetic differentiation with only a minimal gene flow between them. Consistently, in a population structure analysis, the three groups are also clearly distinguished, which is consistent with the results of the population differentiation coefficient. Demographic analyses revealed that the effective population size ( Ne ) of the SCS population underwent a dramatic bottleneck during the Last Glacial Maximum (LGM), followed by a substantial recovery, whereas the RS and AS populations maintained stable Ne values throughout this period. To investigate adaptive responses to climate change in the SCS population, we employed selective elimination analysis, which identified 21 candidate genes potentially involved in environmental adaptation. Of particular significance were myo5a , hps4 , znf385a , msh3 , and pfkfb4 , which likely play crucial roles in the adaptive mechanisms of H. kelloggi . This comprehensive study not only illuminates the genetic diversity patterns of H. kelloggi but also provides a valuable foundation for future investigations into the species' evolutionary adaptations.

Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genome assemblies revealed suggestive gene content differences and provided the opportunity for detailed genetic analyses. We created a single-cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined the spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a , and prdm16 . We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting that derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how the novelties of these fish evolved., Competing Interests: HH, HP, CS, SB, VG, MW, WC No competing interests declared, (© 2024, Healey et al.)

Nucleoredoxin (NXN) is a prominent oxidoreductase enzyme, classified under the thioredoxin family, and plays a pivotal role in regulating cellular redox homeostasis. Although the functional characterization of NXN has been extensively studied in mammals, its role in fish remains relatively unexplored. In this study, the NXN gene from Planiliza haematocheilus (PhNXN) was molecularly and functionally characterized using in silico tools, expression analyses, and in vitro assays. The predicted protein sequence of PhNXN contained 418 amino acids with an anticipated molecular mass of 47.53 kDa. It comprised a highly conserved 188 CPPC 191 catalytic motif in the central NXN domain and two thioredoxin-like domains enriched with conserved Cys residues. PhNXN protein was primarily localized in the cytoplasm and nucleus of the cells. The spatial and temporal expression analyses of PhNXN mRNA showed the highest expression level in the brain under normal physiological conditions, while a significant modulation was detected in the blood and head kidney following immunostimulation with polyinosinic: polycytidylic acid, lipopolysaccharides, and Lactococcus garvieae. Recombinant PhNXN protein exhibited DPPH radical scavenging, thiol-dependent disulfide reduction, and cupric ion reduction activities. Additionally, PhNXN overexpression significantly suppressed oxidative stress-induced cell death, heavy metal cation-induced reactive oxygen species production, and viral hemorrhagic septicemia virus-induced cellular apoptosis in fish cells. Furthermore, PhNXN overexpression in RAW 264.7 cells demonstrated notable nitric oxide scavenging activity, M2-type polarization, and anti-inflammatory effect. Collectively, the overall findings of the study highlight the antioxidant and immunological functions of PhNXN in red-lip mullet., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This case study investigates the seasonal prevalence, morphological characteristics, molecular identity, host immune response, and environmental factors influencing Caligus clemensi infestations in farmed European seabass and flathead grey mullet at a single marine fish farm. A total of 600 fish (400 seabass, 200 mullet) were examined over one year, revealing seasonal fluctuations in parasite prevalence and intensity. C. clemensi infestation peaked in winter for seabass (75 % prevalence) and spring for grey mullet (80 % prevalence), with notable declines in summer and autumn for both species. Morphological analysis confirmed C. clemensi as the causative agent, exhibiting sexual dimorphism and characteristic features including a quadrilateral cephalothorax and disk-shaped lunules. Molecular characterization of the 18S rRNA gene corroborated the morphological identification, with phylogenetic analysis revealing a well-supported monophyletic clade for C. clemensi within the family Caligidae. Quantitative real-time PCR demonstrated significantly elevated interleukin-1β (IL-1β) expression in infected fish, indicating a robust inflammatory response. Grey mullet and seabass exhibited high IL-1β upregulation. Histopathological examination revealed severe gill tissue damage, vascular changes, and hepatic steatosis in infected fish. Water quality analysis identified several parameters exceeding permissible limits, including total suspended solids (4800-5100 mg/L), total dissolved solids (32000 mg/L), sulfates (2300 mg/L), and chlorides (18000 mg/L). These elevated levels, likely associated with nearby oil production and refining activities, created conditions conducive to parasite proliferation. The high total suspended solids might provide substrates for C. clemensi settlement and attachment, while increased dissolved ions could stress fish and reduce their parasite resistance. This multidisciplinary case study revealed complex host-parasite interactions between C. clemensi and farmed marine fish, highlighting the need for improved water quality and targeted parasite control to enhance aquaculture sustainability in the Suez Canal region., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The bluespotted cornetfish (Fistularia commersonii), a Lessepsian sprinter species, is distributed in the inter-tropical zone across the entire Indo-Pacific, ranging from the Tropical Eastern Pacific to the Red Sea. In this study, we achieve assembly of a chromosome-level genome for F. commersonii by harnessing the precision of PacBio HiFi sequencing in conjunction with the sophistication of Hi-C sequencing technologies. The assembly yields a size of 638.75 Mb, achieving a distribution rate of 99.35% across 36 chromosomes. Notably, the contig N50 value stands at 15.67 Mb, while the scaffold N50 value reaches an impressive 18.69 Mb. BUSCO analysis demonstrates a genome completeness of 96.9% for F. commersonii. Repetitive elements constitute approximately 29.61% of the genome's size. A total of 20,961 protein-coding genes are predicted, with 95.97% of them having functional annotations. This high-quality assembly provides crucial resources for future studies on the invasion mechanisms of F. commersonii, as well as for comparative genomics research on seahorses and pipefish., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Three-spotted seahorse (Hippocampi trimaculata) is a unique fish with important economic and medicinal values, and its total chromosome number is potentially quite different from other seahorse species. Herein, we constructed a chromosome-level genome assembly for this special seahorse by integration of MGI short-read, PacBio HiFi long-read and Hi-C sequencing techniques. A 416.57-Mb haplotypic genome assembly was obtained. Subsequently, 99.38% of its scaffold sequences were anchored onto 18 chromosomes, with identification of 29.1% repeat sequences in the assembled genome. Additional karyotype analysis validated the diploid chromosomes of 2n = 36, which are remarkably different from other seahorses' 2n = 42 or 44. The genome completeness (BUSCO score: 96.5%, CEGMA score: 97.87%) confirmed that this chromosome-scale assembly is indeed of high quality. Moreover, a total of 18,712 protein-coding genes were annotated, of which 96.36% could be predicted with functions. Based on construction of a phylogenetic tree, we estimated that Hippocampus and Syngnathoides diverged approximately 50.1 million years ago (Mya). Taken together, our genome data presented in this study provide a valuable genetic resource for numerical chromosome changes and in-depth evolutionary and functional investigations, as well as conservation and molecular breeding of this endangered teleost., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The pituitary gland is a key endocrine gland with various physiological functions including metabolism, growth, and reproduction. It comprises several distinct cell populations that release multiple polypeptide hormones. Although the major endocrine cell types are conserved across taxa, the regulatory mechanisms of gene expression and chromatin organization in specific cell types remain poorly understood. Here, we performed simultaneous profiling of the transcriptome and chromatin landscapes in the pituitary cells of the three-spined stickleback (Gasterosteus aculeatus), which represents a good model for investigating the genetic mechanisms underlying adaptive evolution. We obtained pairwise gene expression and chromatin profiles for 5184 cells under short- and long-day conditions. Using three independent clustering analyses, we identified 16 distinct cell clusters and validated their consistency. These results advance our understanding of the regulatory dynamics in the pituitary gland and provide a reference for future research on comparative physiology and evolutionary biology., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Seahorses represent the only known group of animals with male pregnancy. Seahorses have small testis that produce a limited quantity of sperm. To date, the response of this immune-privileged organ to pathogenic infections has not been reported. β-defensin (BD) is an important innate immune defence factor against pathogens in vertebrate testis. To elucidate its immunoprotection in seahorse testis, we identified the Hippocampus erectus β-defensin (HeBD) sequence in its genome via phylogenetic tree and protein-sequence structure analysis. Gene-expression analysis showed that HeBD was highly expressed in the seahorse testis and was significantly upregulated after bacterial infection, indicating that HeBD expression was related to testicular immune responses. Furthermore, antibacterial activity testing demonstrated that the mature HeBD peptide exhibited broad-spectrum aggregation activity but only moderate antibacterial activity. We found that the mature HeBD mature significantly neutralised bacterial endotoxin activity. In conclusion, our results imply that HeBD serves an immunoprotective role in seahorse testis., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Predators exert a powerful selective force, however, predator avoidance can conflict with other important activities such as attracting mates. Decisions over whether to court mates versus avoiding predators are vital to fitness, yet the mechanistic underpinnings of how animals manage such tradeoffs are poorly understood. Here, we investigate the flexibility of behaviour and gene regulation in response to a tradeoff between avoiding predators (survival) and courting potential mates (reproduction) in three-spined stickleback ( Gasterosteus aculeatus ). We compared behavioural and transcriptomic responses of male sticklebacks faced with a courtship opportunity and cues of a predator simultaneously with the responses of males faced with a courtship opportunity or cues of a predator alone, and found that males behaviourally compromised courtship in favour of predator avoidance when faced with a tradeoff between them. The need to manage this tradeoff elicited dynamic changes in brain gene expression, and sets of functionally connected genes were organized into discrete modules based on co-expression. Additionally, we found that behavioural flexibility in response to tradeoffs corresponded to flexibility in gene regulatory network structure. Combined, these results uncover the coordinated response by the brain to a fundamental ecological tradeoff, providing insight into the structure and function of genetic networks underpinning how animals make fitness-influencing decisions.

Chromosomal inversions can play an important role in divergence and reproductive isolation by building and maintaining distinct allelic combinations between evolutionary lineages. Alternatively, they can take the form of balanced polymorphisms that segregate within populations until one arrangement becomes fixed. Many questions remain about how inversion polymorphisms arise, how they are maintained over the long term, and ultimately, whether and how they contribute to speciation. The long-snouted seahorse (Hippocampus guttulatus) is genetically subdivided into geographic lineages and marine-lagoon ecotypes, with shared structural variation underlying lineage and ecotype divergence. Here, we aim to characterize structural variants and to reconstruct their history and suspected role in ecotype formation. We generated a near chromosome-level genome assembly and described genome-wide patterns of diversity and divergence through the analysis of 112 whole-genome sequences from Atlantic, Mediterranean, and Black Sea populations. By also analysing linked-read sequencing data, we found evidence for two chromosomal inversions that were several megabases in length and showed contrasting allele frequency patterns between lineages and ecotypes across the species range. We reveal that these inversions represent ancient intraspecific polymorphisms, one likely being maintained by divergent selection and the other by pseudo-overdominance. A possible selective coupling between the two inversions was further supported by the absence of specific haplotype combinations and a putative functional interaction between the two inversions in reproduction. Lastly, we detected gene flux eroding divergence between inverted alleles at varying levels for the two inversions, with a likely impact on their dynamics and contribution to divergence and speciation., (© 2024 John Wiley & Sons Ltd.)

The Fasin rainbow fish, scientifically named Melanotaenia fasinensis, is highly prized by aquarium enthusiasts for its vibrant colors and adaptability to artificial aquatic environments. This species is endemic to the karst landscape of the Bird's Head region in Papua, Indonesia, and belongs to the family Melanotaeniidae. Discovered relatively recently in 2010, this species was designated as endangered by the International Union for Conservation of Nature (IUCN) in 2021. However, there is currently insufficient data regarding its phylogenetic position. To address this gap, our study employed next-generation sequencing (NGS) to analyze the entire mitochondrial genome of M. fasinensis. The mitochondrial genome comprises 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes, with a total length of 16,731 base pairs. The base composition of the mitogenome revealed percentages of 27.76% adenine (A), 27.34% thymine (T), 16.15% guanine (G), and 28.75% cytosine (C) residues. Our phylogenetic analysis based on sequence data indicated that all species of the Melanotaeniidae family clustered together on the same branch. Furthermore, the intergeneric and interspecific taxonomic positions were explicit and clear. Phylogenetically, Melanotaeniidae were more closely related to the family Isonidae than to the family Atherinomorus. The phylogenetic position of M. fasinensis was relatively basal within the genus Melanotaenia. This study provides valuable molecular insights for further exploration of the phylogeography and evolutionary history of M. fasinensis and other members of the genus Melanotaenia., Competing Interests: Declarations Ethics approval and consent to participate The study protocol received approval from the Ethical Clearance and Research Permits granted by the Directorate of Research and Innovation, BRIN (number: 070/KE.02/SK/04/2023). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

AbstractIndirect genetic effects (IGEs) exist when there is heritable variation in one organism's ability to alter a second organism's traits. For example, parasites have antigens that can induce a host immune response, as well as disparate strategies to evade or suppress host immunity; among-parasite genetic variation in these antigens generates among-host variation in immune traits. Here, we experimentally show that the cestode parasite Schistocephalus solidus exerts an IGE on an immune trait (peritoneal fibrosis) in its threespine stickleback host: stickleback developed strong fibrosis after exposure to some parasite genotypes but not others. A complication arises during coinfection, when two or more parasite genotypes may impose conflicting IGEs on the same host trait. What parasite-controlled trait will the host express? Will the host trait reflect the more immune-stimulatory parasite genotype or the more immune-evasive genotype? These alternatives can be quantified by estimating the dominance coefficient, as if a coinfected host were a heterozygote. We experimentally estimated the dominance of S. solidus IGEs by coinjecting antigens from different parasite genotypes. Contrary to our a priori hypotheses, coinjected antigens induced an overdominant effect, stronger than either parasite's antigens alone. We present a mathematical model showing that the value of this IGE dominance is biologically important, affecting the evolutionary dynamics of parasites in a density- and frequency-dependent manner. The model indicates that overdominance would be detrimental to immigrants when resident prevalence is high. This combination of experimental data and modeling provides an example of a parasite IGE on host traits and the evolutionary significance of IGE dominance.

Mugil cephalus is a species of considerable interest for aquaculture. As a species that is not sexually dimorphic, when building a brood stock with a balanced sex ratio there is difficulty for identification of sex until the animals are quite mature. When mono-sex populations of this species is desired, as in the case of production of females for "bottarga", considerable resource expenditure could be saved if early sorting of sexes were possible to enable selection of a single sex. A recently described sex-associated loci within the follicle stimulating hormone receptor gene (fshr) was identified using genomic DNA sequencing and shown to contain some non-synonymous mutations wherein the females have a tendency to be homozygous and the males heterozygous. The loci identification method is time-consuming and somewhat expensive. We propose that the method described for the identification of the genetic sex of Mugil cephalus, prior to sexual maturation should be rapid and not require DNA sequencing. In this work, we demonstrate the utilization of one of these fshr mutations within this genetic marker in a PCR-RFLP assay. Using this new method, the loci in question shows 77.8 % partitioning with males and 88.9 % partition with females, as referenced to phenotypic sex characterized by histology, thus confirming the partitioning of the genetic marker as seen previously using DNA sequencing. Future applications of this relatively rapid and inexpensive method could contribute to the production of mono-sex farmed stock and open new opportunities for more efficient broodstock management practices in the species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

A new species of freshwater pipefish, Microphis arrakisae sp. nov., is described from the West Indonesian Islands (Java, Bali and Lombok). This species is morphologically very close to Microphis retzii (Bleeker, 1856), which is found in the eastern Indonesian Islands (Sulawesi, Ceram, Ambon and Papua). However, it can be distinguished by its in vivo coloration. Furthermore, genetic analysis of the partial COI gene (barcoding) indicates that it represents a distinct genetic lineage in the Indonesian region.

Seahorses exhibit the unique characteristic of male pregnancy, which incubates numerous embryos in a brood pouch that plays an essential role in enhancing offspring survivability. The pot-belly seahorse (Hippocampus abdominalis) possesses the largest body size among seahorses and is a significant species in Chinese aquaculture. In this study, we revealed the cytological and morphological characteristics, as well as regulatory mechanisms, throughout the entire brood pouch development in H. abdominalis. The brood pouch originated from the abdominal dermis, extending towards the ventral midline. As the dermal layers thicken, the inner epithelium folds, the stroma loosens, and vascularization occurs, culminating in the formation of the brood pouch. Furthermore, through transcriptomic analysis of brood pouches at various developmental stages, 8 key genes (tgfb3, fgf2, wnt7a, pgf, mycn, tln2, jund, ccn4) closely related to the development of brood pouch were identified in the MAPK, Rap1, TGF-β, and Wnt signaling pathways. These genes were highly expressed in the pseudoplacenta and dermal layers at the newly formed stage as examined by in situ hybridization (ISH). The angiogenesis, densification of collagen fibers, and proliferation of fibroblasts and endothelial cells in seahorse brood pouch formation may be regulated by these genes and pathways. Additionally, the expression of the androgen receptor gene (ar) was significantly upregulated during the formation of the brood pouch, and ISH confirmed the expression of the ar gene in the dermis and pseudoplacenta of the brood pouch, highlighting its role in the developmental process. Androgen and flutamide (androgen receptor antagonist) treatments significantly accelerated the formation of the brood pouch and completely inhibited its occurrence respectively, concomitant to the upregulated expression of differentially expressed genes involved above signaling pathways. These findings demonstrated that formation of the brood pouch is determined by androgen and the androgen receptor activates the above signaling pathways in the brood pouch through the regulation of fgf2, tgfb3, pgf, and wnt7a. Interestingly, androgen even induced the formation of the brood pouch in females. We firstly elucidated the formation of the seahorse brood pouch, demonstrating that androgens and their receptors directly induce the thickening, folding, and vascularization of the abdominal dermal layer into a placenta-like structure through multiple signaling pathways. These findings provide foundational insights to further exploring the evolution of male pregnancy and adaptive convergence in viviparity across vertebrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Acute temperature stress was explored in Hippocampus abdominalis through a comprehensive RNA-seq analysis., Methods: RNA-seq was conducted on 20-day-old H. abdominalis after 24 h of temperature stress. Four experimental conditions were established: a control group (18 °C) and three temperature treatment groups (21, 24, and 27 °C)., Results: Seahorse larvae were found to be unaffected by 21 °C and 24 °C and were able to survive for short periods of time during 24 h of incubation, whereas mortality approached 50% at 27 °C. The sequencing process produced 75.63 Gb of high-quality clean data, with Q20 and Q30 base percentages surpassing 98% and 96%, respectively. A total of 141, 333, and 1598 differentially expressed genes were identified in the 21, 24, and 27 °C groups vs. a control comparison group, respectively. Notably, the number of up-regulated genes was consistently higher than that of down-regulated genes across all comparisons. Gene Ontology functional annotation revealed that differentially expressed genes were predominantly associated with metabolic processes, redox reactions, and biosynthetic functions. In-depth KEGG pathway enrichment analysis demonstrated that down-regulated genes were significantly enriched in pathways related to steroid biosynthesis, terpenoid backbone biosynthesis, spliceosome function, and DNA replication. Up-regulated genes were enriched in pathways associated with the FoxO signaling pathway and mitophagy (animal). The results indicated that temperature stress induced extensive changes in gene expression in H. abdominalis , involving crucial biological processes such as growth, biosynthesis, and energy metabolism., Conclusions: This study provided key molecular mechanisms in the response of H. abdominalis to temperature stress, offering a strong basis for future research aimed at understanding and mitigating the effects of environmental stressors on marine species.

Enteritis caused by Vibrio is a highly die-off disease that severely impeded substantial production in seahorse aquaculture. In the present study, challenged with LD50 of concentration of Vibrio harveyi, a total of 161 of susceptible and 166 of resistant individuals were allocated into binary survival phenotypes, thus, to firstly investigate the genetic architecture by genome-wide association study (GWAS) analysis, as well as to evaluate the feasibility of genomic selection (GS) in enteritis disease resistance trait of the lined seahorse Hippocampus erectus . Results indicated that the heritability for resistance to Vibrio harveyi was estimated to be 0.10. And a set of 10 significant/suggestive SNPs in a multiple chromosomes localization were identified, explaining 7.76% to 13.28% of genetic variance. Associated 82 of candidate genes were clustered into signal transduction, cell proliferation, response of external stress, bacteria defence, and anti-inflammatory processes. Moreover, the potential performance of genomic selection (GS) in application in selective breeding for enteritis disease resistance seahorses was assessed by genomic prediction (GP). In general, the predictive accuracy of the genomic estimated breeding value (GEBV) of BayesC exceeded the rrBLUP, BayesA, RKHS, and SVM models while with no significant difference. And the GWAS-informative SNPs was significantly superior in efficience than random selected markers by comparison of predictive performance on different selection strategies of SNPs. Overall, the genetic basis of enteritis disease resistance trait in the lined seahorse is a polygenic genetic architecture. SNPs associated with the important genes of cathepsin L1-like previously reported with respect to disease resistance are consider as potential molecular markers of genetic breeding. Furthermore, GS approach is an appropriate, effective, and less-cost application in breeding enteritis disease-resistant seahorses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Liu, Shen, Lin and Zhang.)

Pigmentation is an excellent trait to examine patterns of evolutionary change because it is often under natural selection. Benthic and limnetic threespine stickleback (Gasterosteus aculeatus) exhibit distinct pigmentation phenotypes, likely an adaptation to occupation of divergent niches. The genetic architecture of pigmentation in vertebrates appears to be complex. Prior QTL mapping of threespine stickleback pigmentation phenotypes has identified several candidate loci. However-relative to other morphological phenotypes (e.g., spines or lateral plates)-the genetic architecture of threespine stickleback pigmentation remains understudied. Here, we performed QTL mapping for two melanic pigmentation traits (melanophore density and lateral barring) using benthic-limnetic F 2 crosses. The two traits mapped to different chromosomes, suggesting a distinct genetic basis. The resulting QTLs were additive, but explained a relatively small fraction of the total variance (~6%). QTLs maps differed by F 1 family, suggesting variation in genetic architecture or ability to detect loci of small effect. Functional analysis identified enriched pathways for candidate loci. Several of the resulting candidate loci for pigmentation, including three loci in enriched pathways (bco1, sulf1, and tyms) have been previously indicated to affect pigmentation in other vertebrates. These findings add to a growing body of evidence suggesting pigmentation is often polygenic., (© 2024. The Author(s).)

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background : Understanding population genetic structures is crucial for planning and implementing conservation programmes to preserve species' adaptive and evolutionary potential and thus ensure their long-term persistence. The grey mullet ( Mugil cephalus ) is a globally distributed coastal fish. Its populations in waters surrounding Taiwan on the western Pacific fringe are divided into at least two stocks (migratory and residential), but questions remain regarding their genetic divergence and gene flow. Methods and Results : To cast more light on this, allozyme variations at 21 presumptive gene loci of 1217 adult grey mullets from 15 localities in Japan, Taiwan and mainland China, and four gene loci from 1470 juveniles from three localities in Taiwan were used to investigate patterns of genetic variation. The mean expected heterozygosity (He) was 0.128-ranging from 0.031 (Matsu) to 0.442 (Kaoping)-and the mean observed heterozygosity (Ho) was 0.086-ranging from 0.017 (Kaohsiung) to 0.215 (Kaoping). Both AMOVA and the high overall mean FST of 0.252 indicated enormous genetic differentiation among populations and the positive mean value of FIS was 0.328, indicating a deficiency of heterozygotes. PCoA indicated that the samples of M. cephalus could be split into three groups and STRUCTURE analysis showed that all individuals were grouped into three genetic clusters. The results of mutation-drift equilibrium tests did not suggest that the populations experienced any recent genetic bottleneck. The results from all localities in the present investigation showed significant change in the GPI-A genotype frequencies with latitudes-e.g., increases in GPI-A*135/135 homozygote frequencies and GPI-A*100/100 frequencies were highly correlated with latitudinal cline. All migratory populations with the GPI-A genotype were almost exclusively the GPI-A*100/100 homozygote. During the life history of M. cephalus , the GPI-A*100/135 heterozygote frequency significantly decreases with age. Conclusions : Based on these data, we suggest that each GPI-A genotype represents trait combinations of higher fitness in some portions of the environment. Furthermore, the genotypic frequencies change in accordance with life stages, suggesting that selection occurs throughout the life span.

A central goal in evolutionary biology is to understand how different evolutionary processes cause trait change in wild populations. However, quantifying evolutionary change in the wild requires linking trait change to shifts in allele frequencies at causal loci. Nevertheless, datasets that allow for such tests are extremely rare and existing theoretical approaches poorly account for the evolutionary dynamics that likely occur in ecological settings. Using a decade-long integrative phenome-to-genome time-series dataset on wild threespine stickleback ( Gasterosteus aculeatus ), we identified how different modes of selection (directional, episodic, and balancing) drive microevolutionary change in correlated traits over time. Most strikingly, we show that feeding traits changed by as much 25% across 10 generations which was driven by changes in the genetic architecture (i.e., in both genomic breeding values and allele frequencies at genetic loci for feeding traits). Importantly, allele frequencies at genetic loci related to feeding traits changed at a rate greater than expected under drift, suggesting that the observed change was a result of directional selection. Allele frequency dynamics of loci related to swimming traits appeared to be under fluctuating selection evident in periodic population crashes in this system. Our results show that microevolutionary change in a wild population is characterized by different modes of selection acting simultaneously on different traits, which likely has important consequences for the evolution of correlated traits. Our study provides one of the most thorough descriptions to date of how microevolutionary processes result in trait change in a natural population., Competing Interests: Competing interests statement:The authors declare no competing interest.

Crater lake fishes are common evolutionary model systems, with recent studies suggesting a key role for gene flow in promoting rapid adaptation and speciation. However, the study of these young lakes can be complicated by human-mediated extinctions. Museum genomics approaches integrating genetic data from recently extinct species are, therefore, critical to understanding the complex evolutionary histories of these fragile systems. Here, we examine the evolutionary history of an extinct Southern Hemisphere crater lake endemic, the rainbowfish Melanotaenia eachamensis. We undertook a comprehensive sampling of extant rainbowfish populations of the Atherton Tablelands of Australia alongside historical museum material to understand the evolutionary origins of the extinct crater lake population and the dynamics of gene flow across the ecoregion. The extinct crater lake species is genetically distinct from all other nearby populations due to historic introgression between 2 proximate riverine lineages, similar to other prominent crater lake speciation systems, but this historic gene flow has not been sufficient to induce a species flock. Our results suggest that museum genomics approaches can be successfully combined with extant sampling to unravel complex speciation dynamics involving recently extinct species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)

Repeatability of adaptation to similar environments provides opportunity to evaluate the predictability of natural selection. While many studies have investigated gene expression differences between populations adapted to contrasting environments, the role of post-transcriptional processes such as alternative splicing has rarely been evaluated in the context of parallel adaptation. To address the aforementioned knowledge gap, we reanalysed transcriptomic data from three pairs of threespine stickleback (Gasterosteus aculeatus) ecotypes adapted to marine or freshwater environment. First, we identified genes with repeated expression or splicing divergence across ecotype pairs, and compared the genetic architecture and biological processes between parallelly expressed and parallelly spliced loci. Second, we analysed the extent to which parallel adaptation was reflected at gene expression and alternative splicing levels. Finally, we tested how the two axes of transcriptional variation differed in their potential for evolutionary change. Although both repeated differential splicing and differential expression across ecotype pairs showed tendency for parallel divergence, the degree of parallelism was lower for splicing than expression. Furthermore, parallel divergences in splicing and expression were likely to be associated with distinct cis-regulatory genetic variants and functionally unique set of genes. Finally, we found that parallelly spliced genes showed higher nucleotide diversity than parallelly expressed genes, indicating splicing is less susceptible to genetic variation erosion during parallel adaptation. Our results provide novel insight into the role of splicing in parallel adaptation, and underscore the contribution of splicing to the evolutionary potential of wild populations under environmental change., (© 2024 The Author(s). Journal of Animal Ecology © 2024 British Ecological Society.)

Background: Rainbowfish is a clade of colorful freshwater fish. Melanotaenia praecox is a small rainbowfish species with biological characteristics that make it potentially useful as an experimental model species. We anticipate that M. praecox could become a new model used in various fields, such as ecology, evolution, and developmental biology. However, few previous studies have described experimental set-ups needed to understand the molecular and genetic mechanisms within this species., Results: We describe detailed procedures for genetic engineering in the rainbowfish M. praecox. By using these procedures, we successfully demonstrated CRISPR/Cas-mediated knockout and Tol2 transposon-mediated transgenesis in this species. Regarding the CRISPR/Cas system, we disrupted the tyrosinase gene and then showed that injected embryos lacked pigmentation over much of their body. We also demonstrated that a Tol2 construct, including a GFP gene driven by a ubiquitous promoter, was efficiently integrated into the genome of M. praecox embryos., Conclusions: The establishment of procedures for genetic engineering in M. praecox enables investigation of the genetic mechanisms behind a broad range of biological phenomena in this species. Thus, we suggest that M. praecox can be used as a new model species in various experimental biology fields., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Whether populations adapt to similar selection pressures using the same underlying genetic variants depends on population history and the distribution of standing genetic variation at the metapopulation level. Studies of sticklebacks provide a case in point: when colonizing and adapting to freshwater habitats, three-spined sticklebacks (Gasterosteus aculeatus) with high gene flow tend to fix the same adaptive alleles in the same major loci, whereas nine-spined sticklebacks (Pungitius pungitius) with limited gene flow tend to utilize a more heterogeneous set of loci. In accordance with this, we report results of quantitative trait locus (QTL) analyses using a backcross design showing that lateral plate number variation in the western European nine-spined sticklebacks mapped to 3 moderate-effect QTL, contrary to the major-effect QTL in three-spined sticklebacks and different from the 4 QTL previously identified in the eastern European nine-spined sticklebacks. Furthermore, several QTL were identified associated with variation in lateral plate size, and 3 moderate-effect QTL with body size. Together, these findings indicate more heterogenous and polygenic genetic underpinnings of skeletal armour variation in nine-spined than three-spined sticklebacks, indicating limited genetic parallelism underlying armour trait evolution in the family Gasterostidae., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology.)

In mammals, 17-beta hydroxysteroid dehydrogenase 2 (Hsd17b2) enzyme specifically catalyzes the oxidation of the C17 hydroxyl group and efficiently regulates the activities of estrogens and androgens to prevent diseases induced by hormone disorders. However, the functions of the hsd17b2 gene involved in animal sex differentiation are still largely unclear. The ricefield eel ( Monopterus albus ), a protogynous hermaphroditic fish with a small genome size (2 n = 24), is usually used as an ideal model to study the mechanism of sex differentiation in vertebrates. Therefore, in this study, hsd17b2 gene cDNA was cloned and its mRNA expression profiles were determined in the ricefield eel. The cloned cDNA fragment of hsd17b2 was 1230 bp, including an open reading frame of 1107 bp, encoding 368 amino acid residues with conserved catalytic subunits. Moreover, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis showed that hsd17b2 mRNA expressed strongly in the ovaries at early developmental stages, weakly in liver and intestine, and barely in testis and other tissues. In particular, hsd17b2 mRNA expression was found to peak in ovaries of young fish and ovotestis at the early stage, and eventually declined in gonads from the late ovotestis to testis. Likewise, chemical in situ hybridization results indicated that the hsd17b2 mRNA signals were primarily detected in the cytoplasm of oogonia and oocytes at stage I-II, subsequently concentrated in the granulosa cells around the oocytes at stage Ⅲ-Ⅳ, but undetectable in mature oocytes and male germ cells. Intriguingly, in ricefield eel ovaries, hsd17b2 mRNA expression could be significantly reduced by 17β-estradiol (E2) or tamoxifen (17β-estradiol inhibitor, E2I) induction at a low concentration (10 ng/mL) and increased by E2I induction at a high concentration (100 ng/mL). On the other hand, both the melatonin (MT) and flutamide (androgen inhibitor, AI) induction could significantly decrease hsd17b2 mRNA expression in the ovary of ricefield eel. This study provides a clue for demonstrating the mechanism of sexual differentiation in fish. The findings of our study imply that the hsd17b2 gene could be a key regulator in sexual differentiation and modulate sex reversal in the ricefield eel and other hermaphroditic fishes.

The nine-spined stickleback (Pungitius pungitius) has been increasingly used as a model system in studies of local adaptation and sex chromosome evolution but its current reference genome assembly is far from perfect, lacking distinct sex chromosomes. We generated an improved assembly of the nine-spined stickleback reference genome (98.3% BUSCO completeness) with the aid of linked-read mapping. While the new assembly (v8) was of similar size as the earlier version (v7), we were able to assign 4.4 times more contigs to the linkage groups and improve the contiguity of the genome. Moreover, the new assembly contains a ∼22.8 Mb Y-linked scaffold (LG22) consisting mainly of previously assigned X-contigs, putative Y-contigs, putative centromere contigs, and highly repetitive elements. The male individual showed an even mapping depth on LG12 (pseudo X chromosome) and LG22 (Y-linked scaffold) in the segregating sites, suggesting near-pure X and Y representation in the v8 assembly. A total of 26,803 genes were annotated, and about 33% of the assembly was found to consist of repetitive elements. The high proportion of repetitive elements in LG22 (53.10%) suggests it can be difficult to assemble the complete sequence of the species' Y chromosome. Nevertheless, the new assembly is a significant improvement over the previous version and should provide a valuable resource for genomic studies of stickleback fishes., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Gaining insight into the diversity, structure, and metabolic functions of microbial communities is essential for understanding their roles in host health and ecosystem dynamics. However, research on the seahorse-associated microbiome remains limited, despite these threatened fish facing increasing human pressures worldwide. Here, we explored the microbial diversity and metabolic functions of the skin and gut of the tiger tail seahorse ( Hippocampus comes ) and its surrounding environment using shotgun metagenomics and bioinformatics. Members of the Pseudomonadota phylum were dominant in the skin microbiome, whereas Bacteroidota was dominant in the gut. Bacillota, Actinomycetota, and Planctomycetota were also detected in the seahorse-associated microbiome. Statistical analysis revealed significant differences ( P < 0.01) in species diversity between skin and gut microbiomes, with members belonging to the Moraxellaceae family being dominant on the skin and the Bacteroidaceae family in the gut. Moreover, the surrounding environment (water or sediment) did not have a direct effect on the seahorse microbiome composition. The skin microbiome exhibited a higher abundance of functional genes related to energy, lipid, and amino acid metabolism as well as terpenoids and polyketides metabolism, xenobiotics biodegradation, and metabolism compared with the gut. Despite differences among classes, the total abundance of bacteriocins was similar in both gut and skin microbiomes, which is significant in shaping microbial communities due to their antimicrobial properties. A better knowledge of seahorse microbiomes benefits conservation and sustainable aquaculture efforts, offering insights into habitat protection, disease management, and optimizing aquaculture environments, thereby promoting seahorse health and welfare while minimizing environmental impact and enhancing aquaculture sustainability. NEW & NOTEWORTHY To the best of our knowledge, this study represents the first comprehensive examination of the taxonomic and functional patterns of the skin and gut microbiome in the tiger tail seahorse. These findings have the potential to significantly enhance our understanding of the seahorse-associated microbiome, thereby contributing to the prediction and control of bacterial infections in seahorses, which are a leading cause of high mass mortality rates in seahorse aquaculture and other fish species.

Background: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq)., Results: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions., Conclusions: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations., (© 2024. The Author(s).)

Mate choice plays a fundamental role in speciation, yet we know little about the molecular mechanisms that underpin this crucial decision-making process. Stickleback fish differentially adapted to limnetic and benthic habitats are reproductively isolated and females of each species use different male traits to evaluate prospective partners and reject heterospecific males. Here, we integrate behavioural data from a mate choice experiment with gene expression profiles from the brains of females actively deciding whether to mate. We find substantial gene expression variation between limnetic and benthic females, regardless of behavioural context, suggesting general divergence in constitutive gene expression patterns, corresponding to their genetic differentiation. Intriguingly, female gene co-expression modules covary with male display traits but in opposing directions for sympatric populations of the two species, suggesting male displays elicit a dynamic neurogenomic response that reflects known differences in female preferences. Furthermore, we confirm the role of numerous candidate genes previously implicated in female mate choice in other species, suggesting evolutionary tinkering with these conserved molecular processes to generate divergent mate preferences. Taken together, our study adds important new insights to our understanding of the molecular processes underlying female decision-making critical for generating sexual isolation and speciation.

AbstractAdaptation to replicated environmental conditions can be remarkably predictable, suggesting that parallel evolution may be a common feature of adaptive radiation. An open question, however, is how phenotypic variation itself evolves during repeated adaptation. Here, we use a dataset of morphological measurements from 35 populations of threespine stickleback, consisting of 16 parapatric lake-stream pairs and three marine populations, to understand how phenotypic variation has evolved during transitions from marine to freshwater environments and during subsequent diversification across the lake-stream boundary. We find statistical support for divergent phenotypic covariance ( P ) across populations, with most diversification of P occurring among freshwater populations. Despite a close correspondence between within-population phenotypic variation and among-population divergence, we find that variation in P is unrelated to total variation in population means across the set of populations. For lake-stream pairs, we find that theoretical predictions for microevolutionary change can explain more than 30% of divergence in P matrices across the habitat boundary. Together, our results indicate that divergence in variance structure occurs primarily in dimensions of trait space with low phenotypic integration, correlated with disparate lake and stream environments. Our findings illustrate how conserved and divergent features of multivariate variation can underlie adaptive radiation.

Despite deep evolutionary conservation, recombination rates vary greatly across the genome and among individuals, sexes and populations. Yet the impact of this variation on adaptively diverging populations is not well understood. Here we characterized fine-scale recombination landscapes in an adaptively divergent pair of marine and freshwater populations of threespine stickleback from River Tyne, Scotland. Through whole-genome sequencing of large nuclear families, we identified the genomic locations of almost 50,000 crossovers and built recombination maps for marine, freshwater and hybrid individuals at a resolution of 3.8 kb. We used these maps to quantify the factors driving variation in recombination rates. We found strong heterochiasmy between sexes but also differences in recombination rates among ecotypes. Hybrids showed evidence of significant recombination suppression in overall map length and in individual loci. Recombination rates were lower not only within individual marine-freshwater-adaptive loci, but also between loci on the same chromosome, suggesting selection on linked gene 'cassettes'. Through temporal sampling along a natural hybrid zone, we found that recombinants showed traits associated with reduced fitness. Our results support predictions that divergence in cis-acting recombination modifiers, whose functions are disrupted in hybrids, may play an important role in maintaining differences among adaptively diverging populations., (© 2024. The Author(s).)

Seahorses are characterized by unique characteristics such as a male pregnancy reproductive strategy and grasping preferences, which make these species vulnerable to various environmental risks. Zinc (Zn) is one of the most frequently occurring toxic elements in coastal waters; however, little is known about the effect of Zn exposure on seahorses. In the present study, line seahorses (Hippocampus erectus) were exposed to waterborne Zn (0.2 and 1.0 mg/L) and the impact on growth and gonadal development was investigated. Zn exposure induced growth improvement, but also led to gonadal dysfunction in the lined seahorse. Female seahorses exhibited high testosterone levels, immature follicles, and weight increase after Zn exposure, which is the typical characteristics of a polycystic ovary syndrome (PCOS)-like phenotype. Transcriptomic data suggested that the Zn-induced growth promotion resulted from the dysregulated expression of fat accumulation genes. Further investigation of gene expression profiles in the brain, ovaries, and testes indicated that Zn affected the expression of genes involved in growth, immunity, tissue remodeling, and gonadal development by regulating serum steroid hormone levels and androgen receptor expression. This study not only clarifies the complex impact of Zn on seahorses using physiological, histological, and molecular evidence but can also provide new insights into the mechanism underlying PCOS in reproductive-aged women. Moreover, this work demonstrates the risk of the common practice of Zn supplementation in the aquaculture industry as the consequent growth yield may not represent a healthy condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Wild organisms are regularly exposed to a wide range of parasites, requiring the management of an effective immune response while avoiding immunopathology. Currently, our knowledge of immunoparasitology primarily derives from controlled laboratory studies, neglecting the genetic and environmental diversity that contribute to immune phenotypes observed in wild populations. To gain insight into the immunologic variability in natural settings, we examined differences in immune gene expression of two Alaskan stickleback (Gasterosteus aculeatus) populations with varying susceptibility to infection by the cestode Schistocephalus solidus. Between these two populations, we found distinct immune gene expression patterns at the population level in response to infection with fish from the high-infection population displaying signs of parasite-driven immune manipulation. Further, we found significant differences in baseline immune gene profiles between the populations, with uninfected low-infection population fish showing signatures of inflammation compared to uninfected high-infection population fish. These results shed light on divergent responses of wild populations to the same parasite, providing valuable insights into host-parasite interactions in natural ecosystems., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gene regulatory divergence is thought to play an important role in adaptation, yet its extent and underlying mechanisms remain largely elusive for local adaptation with gene flow. Local adaptation is widespread in marine species despite generally high connectivity and is often associated with tightly linked genomic architectures, such as chromosomal inversions. To investigate gene regulatory evolution under gene flow and the role of inversions associated with local adaptation to a steep thermal gradient, we generated RNA-seq data from Atlantic silversides (Menidia menidia) from two locally adapted populations and their F1 hybrids, reared under two temperatures. We found substantial divergence in gene expression and thermal plasticity between populations, with up to 31% of genes being differentially expressed. Reduced thermal plasticity, temperature-dependent gene misexpression, and the disruption of coexpression networks in hybrids point toward a role of regulatory incompatibilities in local adaptation, particularly under colder temperatures. Chromosomal inversions show an accumulation of regulatory incompatibilities but are not consistently enriched for differentially expressed genes. Together, these results suggest that gene regulation can diverge substantially among populations despite gene flow, partly due to the accumulation of temperature-dependent regulatory incompatibilities within inversions., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

Background: The ricefield eel Monopterus albus undergoes a natural sex change from female to male during its life cycle, and previous studies have shown the potential mechanisms of this transition at the transcriptional and protein levels. However, the changes in protein levels have not been fully explored, especially in the intersexual stage., Results: In the present study, the protein expression patterns in the gonadal tissues from five different periods, the ovary (OV), early intersexual stage gonad (IE), middle intersexual stage gonad (IM), late intersexual stage gonad (IL), and testis (TE), were determined by untargeted proteomics sequencing. A total of 5125 proteins and 394 differentially expressed proteins (DEPs) were detected in the gonadal tissues. Of the 394 DEPs, there were 136 between the OV and IE groups, 20 between the IM and IE groups, 179 between the IL and IM groups, and 59 between the TE and IL groups. Three candidate proteins, insulin-like growth factor 2 mRNA-binding protein 3 isoform X1 (Igf2bp3), triosephosphate isomerase (Tpi), and Cu-Zn superoxide dismutase isoform X1 [(Cu-Zn) Sod1], were validated by western blotting to verify the reliability of the data. Furthermore, metal metabolite-related proteins were enriched in the IL vs. IM groups and TE vs. IL groups, which had close relationships with sex change, including Cu 2+ -, Ca 2+ -, Zn 2+ - and Fe 2+ /Fe 3+ -related proteins. Analysis of the combined transcriptome data revealed consistent protein/mRNA expression trends for two metal metabolite-related proteins/genes [LOC109953912 and calcium Binding Protein 39 Like (cab39l)]. Notably, we detected significantly higher levels of Cu 2+ during the sex change process, suggesting that Cu 2+ is a male-related metal metabolite that may have an important function in male reproductive development., Conclusions: In summary, we analyzed the protein profiles of ricefield eel gonadal tissues in five sexual stages (OV, IE, IM, IL, and TE) and verified the plausibility of the data. After preforming the functional enrichment of metal metabolite-related DEPs, we detected the contents of the metal metabolites Zn 2+ , Cu 2+ , Ca 2+ , and Fe 2+ /Fe 3+ at these five stages and screened for (Cu-Zn) Sod1 and Mmp-9 as possible key proteins in the sex reversal process., (© 2024. The Author(s).)

AbstractThe mechanisms underlying the divergence of reproductive strategies between closely related species are still poorly understood. Additionally, it is unclear which selective factors drive the evolution of reproductive behavioral variation and how these traits coevolve, particularly during early divergence. To address these questions, we quantified behavioral differences in a recently diverged pair of Nova Scotian three-spined stickleback ( Gasterosteus aculeatus ) populations, which vary in parental care, with one population displaying paternal care and the other lacking this. We compared both populations, and a full reciprocal F 1 hybrid cross, across four major reproductive stages: territoriality, nesting, courtship, and parenting. We identified significant divergence in a suite of heritable behaviors. Importantly, F 1 hybrids exhibited a mix of behavioral patterns, some of which suggest sex linkage. This system offers fresh insights into the coevolutionary dynamics of reproductive behaviors during early divergence and offers support for the hypothesis that coevolutionary feedback between sexual selection and parental care can drive rapid evolution of reproductive strategies.

When populations repeatedly adapt to similar environments they can evolve similar phenotypes based on shared genetic mechanisms (parallel evolution). The likelihood of parallel evolution is affected by demographic history, as it depends on the standing genetic variation of the source population. The three-spined stickleback ( Gasterosteus aculeatus ) repeatedly colonized and adapted to brackish and freshwater. Most parallel evolution studies in G. aculeatus were conducted at high latitudes, where freshwater populations maintain connectivity to the source marine populations. Here, we analysed southern and northern European marine and freshwater populations to test two hypotheses. First, that southern European freshwater populations (which currently lack connection to marine populations) lost genetic diversity due to bottlenecks and inbreeding compared to their northern counterparts. Second, that the degree of genetic parallelism is higher among northern than southern European freshwater populations, as the latter have been subjected to strong drift due to isolation. The results show that southern populations exhibit lower genetic diversity but a higher degree of genetic parallelism than northern populations. Hence, they confirm the hypothesis that southern populations have lost genetic diversity, but this loss probably happened after they had already adapted to freshwater conditions, explaining the high degree of genetic parallelism in the south.

Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Escobar-Sierra, Lampert. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In Brief: Seahorses exhibit male pregnancy and are thus valuable comparative models for the study of the physiology and evolution of pregnancy. This study shows that protein is transported from fathers to developing embryos during gestation, and provides new knowledge about paternal contributions to embryonic development., Abstract: Syngnathid embryos (seahorses, pipefishes and seadragons) develop on or in the male in a specialised brooding structure (brood pouch). Seahorse brood pouches supply nutrients, including lipids, to developing embryos (patrotrophy). We tested the hypothesis that proteins, vital for gene regulation and tissue growth during embryogenesis, are also transported from father to embryos, using the Australian pot-bellied seahorse, Hippocampus abdominalis. We used dry masses and total nitrogen content to estimate the total protein content of newly fertilised egg and neonate H. abdominalis. Neonates contained significantly greater protein mass than newly fertilised eggs. This result indicates that paternal protein transport to developing embryos occurs during H. abdominalis pregnancy. This study is the first to show paternal protein transport during pregnancy in seahorses, and furthers our understanding of paternal influence on embryonic development in male pregnant vertebrates.

Directly observing the chronology and tempo of adaptation in response to ecological change is rarely possible in natural ecosystems. Sedimentary ancient DNA (sedaDNA) has been shown to be a tractable source of genome-scale data of long-dead organisms 1 , 2 , 3 and to thereby potentially provide an understanding of the evolutionary histories of past populations. 4 , 5 To date, time series of ecosystem biodiversity have been reconstructed from sedaDNA, typically using DNA metabarcoding or shotgun sequence data generated from less than 1 g of sediment. 6 , 7 Here, we maximize sequence coverage by extracting DNA from ∼50× more sediment per sample than the majority of previous studies 1 , 2 , 3 to achieve genotype resolution. From a time series of Late Pleistocene sediments spanning from a marine to freshwater ecosystem, we compare adaptive genotypes reconstructed from the environmental genomes of three-spined stickleback at key time points of this transition. We find a staggered temporal dynamic in which freshwater alleles at known loci of large effect in marine-freshwater divergence of three-spined stickleback (e.g., EDA) 8 were already established during the brackish phase of the formation of the isolation basin. However, marine alleles were still detected across the majority of marine-freshwater divergence-associated loci, even after the complete isolation of the lake from marine ingression. Our retrospective approach to studying adaptation from environmental genomes of three-spined sticklebacks at the end of the last glacial period complements contemporary experimental approaches 9 , 10 , 11 and highlights the untapped potential for retrospective "evolve and resequence" natural experiments using sedaDNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study documents the effect of cryopreservation on motility, DNA integrity, and gene expression in Mugil cephalus sperm. Fresh sperm were cryopreserved using V2 extender (V2E) or 0.3 M glucose, each in combination with one of three cryoprotective agents (CPAs), i.e., 10 % of dimethylsulfoxide, ethylene glycol, or glycerol, all at once. After two different storage (7- vs 60- day) periods in liquid nitrogen, sperm samples were thawed. Single-cell gel electrophoresis was used to detect the DNA integrity. Heat shock proteins (HSPs), HSP70, HSP90 and glutathione peroxidase (GPx2) genes mRNA expression levels was documented using qRT-PCR. The results demonstrated that among 0.3 M glucose + CPAs combinations, EG recorded higher frozen-thawed motility 69 % (7- day) and 59 % (60- day). Similarly, in V2E + CPAs combinations, EG recorded higher frozen-thawed motility 31 % (7- day) and 26 % (60- day). The DNA integrity of all thawed sperm (both periods) did not differ from that of fresh sperm. The qRT-PCR results revealed that in the combination of 0.3 M glucose + CPAs, the level of HSP90 and GPx2 gene expression was found to be upregulated in frozen-thawed sperm on both periods. Whereas, the expression level of the HSP70 gene was down-regulated. On the contrary, in the combination of V2E + CPAs, the expression levels of HSP70, HSP90 and GPx2 genes could not be detected on both periods. Overall, the findings of this study demonstrate that the cryomedium (extender + cryoprotectant) has a more influential role in the motility and levels of gene expression in the frozen-thawed sperm of M. cephalus., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest with this manuscript., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Early life microbial colonizers shape and support the immature vertebrate immune system. Microbial colonization relies on the vertical route via parental provisioning and the horizontal route via environmental contribution. Vertical transmission is mostly a maternal trait making it hard to determine the source of microbial colonization in order to gain insight into the establishment of the microbial community during crucial development stages. The evolution of unique male pregnancy in pipefishes and seahorses enables the disentanglement of both horizontal and vertical transmission, but also facilitates the differentiation of maternal versus paternal provisioning ranging from egg development, to male pregnancy and early juvenile development. Using 16S rRNA amplicon sequencing and source-tracker analyses, we revealed how the distinct origins of transmission (maternal, paternal and horizontal) shaped the juvenile internal and external microbiome establishment in the broad-nosed pipefish Syngnathus typhle . Our data suggest that transovarial maternal microbial contribution influences the establishment of the juvenile gut microbiome whereas paternal provisioning mainly shapes the juvenile external microbiome. The identification of juvenile key microbes reveals crucial temporal shifts in microbial development and enhances our understanding of microbial transmission routes, colonization dynamics and their impact on lifestyle evolution.

Advances in genomic studies have revealed that hybridization in nature is pervasive and raised questions about the dynamics of different genetic and evolutionary factors following the initial hybridization event. While recent research has proposed that the genomic outcomes of hybridization might be predictable to some extent, many uncertainties remain. With comprehensive whole-genome sequence data, we investigated the genetic introgression between 2 divergent lineages of 9-spined sticklebacks (Pungitius pungitius) in the Baltic Sea. We found that the intensity and direction of selection on the introgressed variation has varied across different genomic elements: while functionally important regions displayed reduced rates of introgression, promoter regions showed enrichment. Despite the general trend of negative selection, we identified specific genomic regions that were enriched for introgressed variants, and within these regions, we detected footprints of selection, indicating adaptive introgression. Geographically, we found the selection against the functional changes to be strongest in the vicinity of the secondary contact zone and weaken as a function of distance from the initial contact. Altogether, the results suggest that the stabilization of introgressed variation in the genomes is a complex, multistage process involving both negative and positive selection. In spite of the predominance of negative selection against introgressed variants, we also found evidence for adaptive introgression variants likely associated with adaptation to Baltic Sea environmental conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

AbstractOrganismal divergence can be driven by differential resource use and adaptation to different trophic niches. Variation in diet is a major factor shaping the gut microbiota, which is crucial for many aspects of their hosts' biology. However, it remains largely unknown how host diet diversity affects the gut microbiota, and it could be hypothesized that trophic niche width is positively associated with gut microbiota diversity. To test this idea, we sequenced the 16S ribosomal RNA gene from intestinal tissue of 14 threespine stickleback populations from lakes of varying size on Vancouver Island, Canada, that have been shown to differ in trophic niche width. Using lake size as a proxy for trophic ecology, we found evidence for higher gut microbiota uniqueness among individuals from populations with broader trophic niches. While these results suggest that diet diversity might promote gut microbiota diversity, additional work investigating diet and gut microbiota variation of the same host organisms will be necessary. Yet our results motivate the question of how host population diversity (e.g., ecological, morphological, genetic) might interact with the gut microbiota during the adaptation to ecological niches.

Light is a strong stimulus for the sensory and endocrine systems. The opsins constitute a large family of proteins that can respond to specific light wavelengths. Hippocampus reidi is a near-threatened seahorse that has a diverse color pattern and sexual dimorphism. Over the years, H. reidi's unique characteristics, coupled with its high demand and over-exploitation for the aquarium trade, have raised concerns about its conservation, primarily due to their significant impact on wild populations. Here, we characterized chromatophore types in juvenile and adult H. reidi in captivity, and the effects of specific light wavelengths with the same irradiance (1.20 mW/cm 2 ) on color change, growth, and survival rate. The xanthophores and melanophores were the major components of H. reidi pigmentation with differences in density and distribution between life stages and sexes. In the eye and skin of juveniles, the yellow (585 nm) wavelength induced a substantial increase in melanin levels compared to the individuals kept under white light (WL), blue (442 nm), or red (650 nm) wavelengths. In addition, blue and yellow wavelengths led to a higher juvenile mortality rate in comparison to the other treatments. Adult seahorses showed a rhythmic color change over 24 h, the highest reflectance values were obtained in the light phase, representing a daytime skin lightening for individuals under WL, blue and yellow wavelength, with changes in the acrophase. The yellow wavelength was more effective on juvenile seahorse pigmentation, while the blue wavelength exerted a stronger effect on the regulation of adult physiological color change. Dramatic changes in the opsin mRNA levels were life stage-dependent, which may infer ontogenetic opsin functions throughout seahorses' development. Exposure to specific wavelengths differentially affected the opsins mRNA levels in the skin and eyes of juveniles. In the juveniles, skin transcripts of visual (rh1, rh2, and lws) and non-visual opsins (opn3 and opn4x) were higher in individuals under yellow light. While in the juvenile's eyes, only rh1 and rh2 had increased transcripts influenced by yellow light; the lws and opn3 mRNA levels were higher in juveniles' eyes under WL. Prolonged exposure to yellow wavelength stimulates a robust increase in the antioxidant enzymes sod1 and sod2 mRNA levels. Our findings indicate that changes in the visible light spectrum alter physiological processes at different stages of life in H. reidi and may serve as the basis for a broader discussion about the implications of artificial light for aquatic species in captivity., Competing Interests: Declaration of Competing Interest This manuscript has not been published elsewhere and is not under consideration by any other publisher. The manuscript has been approved by the authors and relevant authorities needed. This article will not be submitted to any other publisher if accepted with this journal. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Allele-specific gene expression evolves rapidly on heteromorphic sex chromosomes. Over time, the accumulation of mutations on the Y chromosome leads to widespread loss of gametolog expression, relative to the X chromosome. It remains unclear if expression evolution on degrading Y chromosomes is primarily driven by mutations that accumulate through processes of selective interference, or if positive selection can also favor the down-regulation of coding regions on the Y chromosome that contain deleterious mutations. Identifying the relative rates of cis-regulatory sequence evolution across Y chromosomes has been challenging due to the limited number of reference assemblies. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an excellent model to identify how regulatory mutations accumulate on Y chromosomes due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across 3 differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions on the Y chromosome exhibited elevated substitution rates and decreased polymorphism when compared to nonfunctional sites, like intergenic regions and synonymous sites. This suggests that many cis-regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked cis-regulatory regions exhibit signs of positive selection quickly after the suppression of recombination and allow comparisons with recent theoretical models that suggest the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome., Competing Interests: Conflict of Interest The authors declare no conflicts of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)