Deciphering dynamic interactions between spermatozoa and the ovarian microenvironment through integrated multi-omics approaches in viviparous black rockfish (Sebastes schlegelii).

The ovarian microenvironment plays a critical role in ensuring the reproductive success of viviparous teleosts. However, the molecular mechanism underlying the interaction between spermatozoa and the ovarian microenvironment has remained elusive. This study aimed to contribute to a better understanding to this process in black rockfish (Sebastes schlegelii) utilizing integrated multi-omics approaches. The results demonstrated significant upregulation of ovarian complement-related proteins and pattern recognition receptors, along with remodeling of glycans on the surface of spermatozoa at early spermatozoa-storage stage (one month after mating). As spermatozoa were stored over time, ovarian complement proteins were progressively repressed by tryptophan and hippurate, indicating a remarkable adaptation of spermatozoa to the ovarian microenvironment. Near fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promoted spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism influencing the dynamics of spermatozoa in the female reproductive tract, providing valuable insights into the molecular networks regulating spermatozoa adaptation and survival in species with internal fertilization.

Semen promotes oocyte development in Sebastesschlegelii elucidating ovarian development dynamics in live-bearing fish.

In some vertebrates and invertebrates, semen release factors affecting female physiology and behavior. Here, we report that semen delivered to females is potentially beneficial for promoting oocyte development in a viviparous teleost, Sebastes schlegelii. 88% of mated ovaries develop normally and give birth to larval fish, whereas 61% of non-mated ovaries are arrested in the previtellogenic stage. Semen's significant role (p < 0.0001) in promoting oocyte development may involve remodeling follicular cells and regulating the expression of the extracellular matrix, which facilitates cell communication. Furthermore, the ovarian response to semen may influence the brain, affecting hormone release, follicular cell development and steroid production, and crucial for oocyte growth. This mechanism, which could potentially delay maternal investment in offspring until male genetic input occurs to avoid energy wastage, has not been previously described in teleosts. These findings enhance our understanding of ovarian development in viviparous fish, with broader implications for reproductive biology.

Dissecting the dynamic cellular transcriptional atlas of adult teleost testis development throughout the annual reproductive cycle.

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species.

Chronic low salinity stress rescued masculinization effect in farmed Cynoglossus semilaevis population.

Salinity, being an indispensable abiotic factor crucial for the survival of marine organisms, has demonstrated diverse alterations globally in response to the current trend of global warming. In this study, the effect of chronic low salinity stress on teleosts' sex differentiation was investigated using Cynoglossus semilaevis, an economically important fish with both genetic and environmental sex determination system. The cultivation experiment was conducted employing artificially simulated seawater of 20 ppt and ambient sea water of 30 ppt to rear juveniles C. semilaevis. Throughout the experiment, the growth performance was assessed and the histology of gonadal development was examined, a significantly lower masculinization rate was observed in LS group. To gain further insights, transcriptome analysis was conducted using raw reads obtained from 53 libraries derived from gonads of 55 days post fertilization (dpf) and 100 dpf juveniles in both LS and CT groups. GO/KEGG enrichment were further proceeded, Terms and pathways involved in reproduction ability, germ cell proliferation, immune function, steroid metabolism etc., were illuminated and a possible crosstalk between HPI and HPG axis was proposed. WGCNA was conducted and two hub genes, hspb8-like and Histone H2A.V were exhibited to be of great significance in the changes of masculinization rate. Our findings provided solid reference for sex differentiation study of GSD + ESD species in a constantly changing ocean environment, as well as practice guiding significance for the environmental management for the culture of C. semilaevis.

Chromosome-level genome assembly of humpback grouper using PacBio HiFi reads and Hi-C technologies.

The humpback grouper (Cromileptes altivelis), a medium-sized coral reef teleost, is a naturally rare species distributed in the tropical waters of the Indian and Pacific Oceans. It has high market value, but artificial reproduction and breeding remain limited and need to be improved. Here, we assembled the genome with 1.08 Gb, with a contig N50 of 43.78 Mb. A total of 96.59% of the assembly anchored to 24 pseudochromosomes using Hi-C technology. It contained 24,442 protein-coding sequences, of which 99.3% were functionally annotated. The completeness of the assembly was estimated to be 97.3% using BUSCO. The phylogenomic analysis suggested that humpback grouper should be classified into the genus Epinephelus rather than Cromileptes. The comparative genomic analysis revealed that the gene families related to circadian entrainment were significantly expanded. The high-quality reference genome provides useful genomic tools for exploiting the genomic resource of humpback grouper and supports the functional genomic study of this species in the future.

Comparative Studies on Duplicated foxl2 Paralogs in Spotted Knifejaw Oplegnathus punctatus Show Functional Diversification.

As a member of the forkhead box L gene family, foxl2 plays a significant role in gonadal development and the regulation of reproduction. During the evolution of deuterostome, whole genome duplication (WGD)-enriched lineage diversifications and regulation mechanisms occurs. However, only limited research exists on foxl2 duplication in teleost or other vertebrate species. In this study, two foxl2 paralogs, foxl2 and foxl2l, were identified in the transcriptome of spotted knifejaw (Oplegnathus punctatus), which had varying expressions in the gonads. The foxl2 was expressed higher in the ovary, while foxl2l was expressed higher in the testis. Phylogenetic reconstruction, synteny analysis, and the molecular evolution test confirmed that foxl2 and foxl2l likely originated from the first two WGD. The expression patterns test using qRT-PCR and ISH as well as motif scan analysis revealed evidence of potentially functional divergence between the foxl2 and foxl2l paralogs in spotted knifejaw. Our results indicate that foxl2 and foxl2l may originate from the first two WGD, be active in transcription, and have undergone functional divergence. These results shed new light on the evolutionary trajectories of foxl2 and foxl2l and highlights the need for further detailed functional analysis of these two duplicated paralogs.

Investigation of Gene Networks in Three Components of Immune System Provides Novel Insights into Immune Response Mechanisms against Edwardsiella tarda Infection in Paralichthys olivaceus.

As a quintessential marine teleost, Paralichthys olivaceus demonstrates vulnerability to a range of pathogens. Long-term infection with Edwardsiella tarda significantly inhibits fish growth and even induces death. Gills, blood, and kidneys, pivotal components of the immune system in teleosts, elicit vital regulatory roles in immune response processes including immune cell differentiation, diseased cell clearance, and other immunity-related mechanisms. This study entailed infecting P. olivaceus with E. tarda for 48 h and examining transcriptome data from the three components at 0, 8, and 48 h post-infection employing weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis. Network analyses revealed a series of immune response processes after infection and identified multiple key modules and key, core, and hub genes including xpo1, src, tlr13, stat1, and mefv. By innovatively amalgamating WGCNA and PPI network methodologies, our investigation facilitated an in-depth examination of immune response mechanisms within three significant P. olivaceus components post-E. tarda infection. Our results provided valuable genetic resources for understanding immunity in P. olivaceus immune-related components and assisted us in further exploring the molecular mechanisms of E. tarda infection in teleosts.

Immunological characterization and function analysis of L-type lectin from spotted knifejaw, Oplegnathus punctatus.

Lily-type lectin (LTL) plays significant roles in innate immune response against pathogen infection. LTL in animals and plants has received widespread attention. In the present study, an LTL (OppLTL) was identified from spotted knifejaw Oplegnathus punctatus. The OppLTL encoded a typical Ca2+-dependent carbohydrate-binding protein containing a CRD domain. The qRT-PCR showed that it was mainly expressed in the gill and was significantly upregulated after Vibrio anguillarum challenge. The agglutination analysis showed that the recombinant OppLTL could bind and agglutinate Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. However, the binding activity was different. Meanwhile, the recombinant OppLTL could hemagglutinate mammalian and teleost erythrocytes. Subcellular localization revealed that OppLTL was mainly detected in the cytoplasm of HEK293T cells. The dual-luciferase analysis revealed that OppLTL could inhibit the activity of the NF-κB signal pathway in HEK293T cells after OppLTL overexpression. These findings collectively demonstrated that OppLTL could be involved in host innate immune response and defense against bacterial infection in spotted knifejaw.

Transcriptome and Network Analyses Reveal the Gene Set Involved in PST Accumulation and Responses to Toxic Alexandrium minutum Exposure in the Gills of Chlamys farreri.

Bivalve molluscs are filter-feeding organisms that can accumulate paralytic shellfish toxins (PST) through ingesting toxic marine dinoflagellates. While the effects of PST accumulation upon the physiology of bivalves have been documented, the underlying molecular mechanism remains poorly understood. In this study, transcriptomic analysis was performed in the gills of Zhikong scallop (Chlamys farreri) after 1, 3, 5, 10, and 15 day(s) exposure of PST-producing dinoflagellate Alexandrium minutum. Higher numbers of differentially expressed genes (DEGs) were detected at day 1 (1538) and day 15 (989) than that at day 3 (77), day 5 (82), and day 10 (80) after exposure, and most of the DEGs were only regulated at day 1 or day 15, highlighting different response mechanisms of scallop to PST-producing dinoflagellate at different stages of exposure. Functional enrichment results suggested that PST exposure induced the alterations of nervous system development processes and the activation of xenobiotic metabolism and substance transport processes at the acute and chronic stages of exposure, respectively, while the immune functions were inhibited by PST and might ultimately cause the activation of apoptosis. Furthermore, a weighted gene co-expression network was constructed, and ten responsive modules for toxic algae exposure were identified, among which the yellow module was found to be significantly correlated with PST content. Most of the hub genes in the yellow module were annotated as solute carriers (SLCs) with eight being OCTN1s, implying their dominant roles in regulating PST accumulation in scallop gills. Overall, our results reveal the gene set responding to and involved in PST accumulation in scallop gills, which will deepen our understanding of the molecular mechanism of bivalve resistance to PST.

Identification and functional characterization of Pomstna in Japanese flounder (Paralichthys olivaceus).

Myostatin (MSTN) as a negative regulator of muscle growth has been identified in Japanese flounder. Yet, most fish experienced the teleost specific genome duplication and possess at least two mstn genes. In current study, the second mstn gene named Pomstna is identified in Japanese flounder. Pomstna is clustered with other mstn2 of teleosts and owned highly conserved TGF-beta domain. In addition to muscle, Pomstna also highly expressed in brain and spleen. Using the primarily cultured muscle cells of Japanese flounder, we found that Pomstna could inhibit the proliferation and differentiation of muscle cells in vitro. As a ligand of TGF-beta signaling pathway, Pomstnb could regulate the expression of p21 and myod by activating the TGF-beta signaling pathway. Different from the function of Pomstnb, Pomstna could not activate the TGF-beta signaling pathway in vitro. During the differentiation of PoM cells, the expression of Pomstnb decreased significantly but the expression of Pomstna showed no change. Our study suggests that Pomstna could negatively regulate the growth and differentiation of muscle like Pomstnb yet through a different regulatory mechanism than Pomstnb. The present study suggests that muscle proliferation and differentiation were regulated by mstn not only through the TGF-beta signaling pathway but also other unknown mechanisms.

Genome-wide identification, characterization and expression profiling of TRAF family genes in Sebastes schlegelii.

Tumor necrosis factor receptor-associated factors (TRAFs) are signaling mediators for Toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily that play important roles in organism immune response. However, reports on systematic identification of TRAF gene family in teleost fish and the function of TRAFs in innate immunity of black rockfish (Sebastes schlegelii) are lacked. In our study, eight TRAF genes were identified and characterized, namely, SsTRAF2a, SsTRAF2a-like, SsTRAF2b, SsTRAF3, SsTRAF4, SsTRAF5, SsTRAF6 and SsTRAF7 in S. schegelii. Furthermore, we analyzed their sequences, conserved domains, gene structures, motif compositions, phylogeny, tissue expression patterns in healthy and Vibro. anguillarum challenged individuals. All the SsTRAFs contained typical conserved domain, including C-terminal MATH domain and N-terminal RING finger domain. Analyses of gene structures and motifs showed the distribution of exon-intron and conserved motifs in S. schegelii and serval other teleost fish. We also analyzed the expression file of SsTRAFs in five immune-relate organs, liver, spleen, kidney, gill and intestine in healthy and bacterial challenged fish. The results indicated that all SsTRAF member were widely involved in immune response after pathogenic bacteria infection. In summary, the analyses of TRAFs in S. schegelii will be helpful to better understand the diverse roles of TRAF genes in the innate immune response to bacterial challenge.

Molecular Mechanism Based on Histopathology, Antioxidant System and Transcriptomic Profiles in Heat Stress Response in the Gills of Japanese Flounder.

As an economically important flatfish in Asia, Japanese flounder is threatened by continuously rising temperatures due to global warming. To understand the molecular responses of this species to temperature stress, adult Japanese flounder individuals were treated with two kinds of heat stress-a gradual temperature rise (GTR) and an abrupt temperature rise (ATR)-in aquaria under experimental conditions. Changes in histopathology, programmed cell death levels and the oxidative stress status of gills were investigated. Histopathology showed that the damage caused by ATR stress was more serious. TUNEL signals confirmed this result, showing more programmed cell death in the ATR group. In addition, reactive oxygen species (ROS) levels and the 8-O-hDG contents of both the GTR and ATR groups increased significantly, and the total superoxide dismutase (T-SOD) activities and total antioxidant capacity (T-AOC) levels decreased in the two stressed groups, which showed damage to antioxidant systems. Meanwhile, RNA-seq was utilized to illustrate the molecular mechanisms underyling gill damage. Compared to the control group of 18 °C, 507 differentially expressed genes (DEGs) were screened in the GTR group; 341 were up-regulated and 166 were down-regulated, and pathway enrichment analysis indicated that they were involved in regulation and adaptation, including chaperone and folding catalyst pathways, the mitogen-activated protein kinase signaling (MAPK) pathway and DNA replication protein pathways. After ATR stress, 1070 DEGs were identified, 627 were up-regulated and 423 were down-regulated, and most DEGs were involved in chaperone and folding catalyst and DNA-related pathways, such as DNA replication proteins and nucleotide excision repair. The annotation of DEGs showed the great importance of heat shock proteins (HSPs) in protecting Japanese flounder from heat stress injury; 12 hsp genes were found after GTR, while 5 hsp genes were found after ATR. In summary, our study records gill dysfunction after heat stress, with different response patterns observed in the two experimental designs; chaperones were activated to defend heat stress after GTR, while replication was almost abandoned due to the severe damage consequent on ATR stress.

Genome-wide identification and analysis of scavenger receptors and their expression profiling in response to Edwardsiella tarda infection in Japanese flounder (Paralichthys olivaceus).

The scavenger receptors (SRs) gene family, as one of pattern recognition receptors, participates in the innate immune response in diverse lineages. However, the systematic identification, characteristics and functions of SRs family are lacking in teleost. Here, we identified all 19 SRs family members in Japanese flounder (Paralichthys olivaceus) based on the genome and transcriptome data. Phylogenetic and Ka/Ks analysis demonstrated that these SRs genes were divided into five classes and all exhibited pronounced purified selection pressures. Whole genome duplication event was found in colec12, scarb2, and lamp1. Gene structure, functional domain and motif distribution analyses indicated that SRs within the different subfamilies are severely conservative. SRs genes showed diverse expression patterns in the embryogenesis and unchanged tissues. The regulations of 14 SRs genes in blood, gill and kidney after E. tarda infection suggested their roles in innate immune response. Meanwhile, ten SRs genes were differentially expressed after E. tarda stimulation in macrophages in vitro. Then we proved that PoSCARA3 could suppress the activity of NF-κB and AP-1 in HEK 293T cells by dual-luciferase assays. In summary, this study provided valuable basis for further functional characterization and immune functions of SRs genes in P. olivaceus.

Genomic and Transcriptomic Landscape and Evolutionary Dynamics of Heat Shock Proteins in Spotted Sea Bass (Lateolabrax maculatus) under Salinity Change and Alkalinity Stress.

The heat shock protein (Hsp) superfamily has received accumulated attention because it is ubiquitous and conserved in almost all living organisms and is involved in a wide spectrum of cellular responses against diverse environmental stresses. However, our knowledge about the Hsp co-chaperon network is still limited in non-model organisms. In this study, we provided the systematic analysis of 95 Hsp genes (LmHsps) in the genome of spotted sea bass (Lateolabrax maculatus), an important aquaculture species in China that can widely adapt to diverse salinities from fresh to sea water, and moderately adapt to high alkaline water. Through in silico analysis using transcriptome and genome database, we determined the expression profiles of LmHsps in response to salinity change and alkalinity stress in L. maculatus gills. The results revealed that LmHsps were sensitive in response to alkalinity stress, and the LmHsp40-70-90 members were more actively regulated than other LmHsps and may also be coordinately interacted as co-chaperons. This was in accordance with the fact that members of LmHsp40, LmHsp70, and LmHsp90 evolved more rapidly in L. maculatus than other teleost lineages with positively selected sites detected in their functional domains. Our results revealed the diverse and cooperated regulation of LmHsps under alkaline stress, which may have arisen through the functional divergence and adaptive recruitment of the Hsp40-70-90 co-chaperons and will provide vital insights for the development of L. maculatus cultivation in alkaline water.

Intensive masculinization caused by chronic heat stress in juvenile Cynoglossus semilaevis: Growth performance, gonadal histology and gene responses.

The sea temperature has been observed to chronically increase during the past decades, leaving unpredictable influences to the marine biological resources. Thus, it is of vital significance to study the biological responses of ocean inhabited organisms with the artificially stimulated heat stress environment. Cynoglossus semilaevis provides us with an ideal model to study the influence of chronic heat stress on the sexual differentiation in marine teleosts for its genetic sex determination (GSD) + environmental effected (EE) sex determination system. In this study, the comparative experiment was conducted employing heated seawater (HT group) and ambient seawater (CT group) to cultivate juvenile C. semilaevis respectively. Significant differences were exhibited in growth performance and a delayed germ cell development effect was found in pseudomales formed under chronic heat stress. Using transcriptome analysis, the transcription profile of 55 days post fertilization (dpf) and 100 dpf juveniles' gonads were studied. A total of 47 libraries were constructed with an average mapping rate of 94.63% after assembling. GO and KEGG enrichment were proceeded using DEGs screened out between (1) pseudomale gonads at 55 dpf and 100 dpf in HT and CT group (2) pseudomale and female gonads at 55 dpf and 100 dpf in HT and CT group. Terms and pathways involved in steroid stimulation, reproduction ability, germ cell proliferation et al. were shed light on. The expression pattern of 29 DEGs including amh, hsp90b1, pgr et al. were also provided to supplement the results of functional enrichment. Weighted gene co-expression networks analysis (WGCNA) was constructed and hspb8-like, histone H2A.V were exhibited to play vital roles in the heat-induced masculinization. Our findings facilitate the understanding for transcriptional variations in intensive masculinization cause by chronic heat stress of C. semilaevis and provide referable study of the influences on the teleosts in elevated sea temperature.

Transcriptomic Analysis Reveals Functional Interaction of mRNA-lncRNA-miRNA in Steroidogenesis and Spermatogenesis of Gynogenetic Japanese Flounder (Paralichthys olivaceus).

Teleost fishes exhibit extraordinary diversity, plasticity and adaptability with their sex determination and sexual development, and there is growing evidence that non-coding RNAs (ncRNAs) are emerging as critical regulators of reproduction. Japanese flounder (Paralichthys olivaceus) is an important marine cultured fish that presents significant sexual dimorphism with bigger females, in which gynogenesis has been applied for aquaculture industry. In order to reveal the regulatory mechanisms of sexual development in gynogenetic female and sex-reversed neo-male P. olivaceus, the lncRNA-miRNA-mRNA interactions were investigated using high-throughput sequencing. A total of 6772 differentially expressed mRNAs (DEmRNAs), 2284 DElncRNAs, and 244 DEmiRNAs were obtained between gynogenetic female ovaries and sex-reversed neo-male testes. Genes in the steroid hormone biosynthesis and secretion pathway were enriched and mostly significantly upregulated in neo-male testes. Subsequently, network analysis uncovered high functional specificity for gynogenetic P. olivaceus sperm motility, as co-expressed DEmRNAs were significantly enriched in microtubule and cytoskeleton-related biological processes. Clustered miRNAs were characterized in the P. olivaceus genome with examples of the largest conserved let-7 clusters. The 20 let-7 members are distributed in 11 clusters and may not transcribe together with their neighboring miR-125b, with let-7 repressing cyp11a and miR-125b repressing esr2b, both as key steroidogenesis pathway genes. In summary, this study provides comprehensive insights into the mRNA-miRNA-lncRNA functional crosstalk in teleost sexual development and gametogenesis and will expand our understanding of ncRNA biology in teleost gynogenesis.

Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers.

MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (Paralichthys olivaceus), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (miR-34a-5p, miR-205-5p, miR-101a-3p, miR-22-3p, miR-23a-3p, miR-210-5p, miR-30c-5p, U6, 5S rRNA, and 18S rRNA) were chosen as potential references to test their expression stability among P. olivaceus tissues, and in livers of P. olivaceus infected with Edwardsiella tarda at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, miR-22-3p and miR-23a-3p were proved to be the suitable combination as reference miRNAs for both P. olivaceus normal tissues and livers infected with E. tarda, and they were successfully applied to normalize miR-7a and miR-221-5p expression in P. olivaceus livers in response to E. tarda infection. All these results provide valuable information for P. olivaceus miRNA quantitative expression analysis in the future.

Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus).

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.

Evolutionary dynamics and conserved function of the Tudor domain-containing (TDRD) proteins in teleost fish.

Tudor domain-containing (TDRD) proteins, the germline enriched protein family, play essential roles in the process of gametogenesis and genome stability through their interaction with the PIWI-interacting RNA (piRNA) pathway. Several studies have suggested the rapid evolution of the piRNA pathway in teleost lineages with striking reproductive diversity. However, there is still limited information about the function and evolution of Tdrd genes in teleost species. In this study, through genome wide screening, 13 Tdrd family genes were identified in economically important aquaculture fish, including spotted sea bass (Lateolabrax maculatus), Asian sea bass (Lates calcarifer), and tongue sole (Cynoglossus semilaevis). With copy number, structure, phylogeny, and synteny analysis, duplication of Tdrd6 and Tdrd7, as well as loss of Stk31 and Tdrd10, were characterized in teleost lineages. Codon based molecular evolution analysis indicated faster evolution of teleost Tdrd genes than that in mammals, potentially associated with the accelerated evolution of the piRNA pathway in teleost lineages. The evolutionary diversity of Tdrd genes was also detected between different teleost lineages. RNA-seq analysis showed that most teleost Tdrd genes were dominantly expressed in gonads, particularly highly expressed in testis, such as Tdrd6, Tdrd7a, Tdrd9, Ecat8, and Tdrd15. The varied expression and evolutionary pattern between the duplicated Tdrd6 and Tdrd7 in teleosts may indicate their functional diversification. All these results suggest a conserved function of teleost Tdrd family in gametogenesis and the piRNA pathway, which could lay a foundation for the evolution of Tdrd genes and be helpful for further deciphering of Tdrd functions in teleosts. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-021-00118-7.

Identification, evolution and expression analyses of mapk gene family in Japanese flounder (Paralichthys olivaceus) provide insight into its divergent functions on biotic and abiotic stresses response.

Mitogen-activated protein kinase (MAPK) are a series of serine/threonine protein kinases showing evolutionary conservation, which can be activated by many stimulus signals and then transfer them from cell membrane to nucleus. MAPKs regulate a variety of biological processes, such as apoptosis, hormone signaling and immune response. In this study, 14 putative mapk genes in Japanese flounder were identified, and their basic physical and chemical properties were characterized. Phylogenetic analysis showed that mapk genes were divided into three main subfamilies, including ERK, JNK and the p38 MAPK. Selection pressure analysis revealed they were evolutionarily-constrained and undergone strong purifying selection. Gene structure and conserved protein motif comparison suggested high levels of conservation in members of mapk gene family. The expression patterns were further investigated in each embryonic and larval development stages and different tissues. In addition, RNA-seq analyses after bacteria and temperature stresses suggested mapk genes had different expression patterns. Three mapk genes showed significant differences in response to E. tarda challenge and five were induced significantly after temperature stress, indicating their potential functions. This systematic analysis provided valuable information for further understanding of the regulation mechanism of mapk gene family under different stresses in Japanese flounder.