Comparative mitochondrial genome analysis provides new insights into the classification of Modiolinae.

BACKGROUND: Mitochondrial genomes have become a powerful tool for studying molecular genetics and phylogeny of mollusks. Currently, the position of Modiolinae within Mytilidae and the taxonomic and phylogenetic relationships within Modiolinae were still controversial. This study focuses on the complete mitochondrial genomes of two species: Modiolus modulaides (Röding, 1798) and Modiolus auriculatus Krauss, 1848, which have not been sequenced before. METHODS AND RESULTS: We assembled and characterized the mitochondrial genomes of M. modulaides and M. auriculatus and then analyzed the phylogenetic relationships. The mitochondrial genomes of M. modulaides and M. auriculatus were 15,422 bp and 16,027 bp, respectively. Both of them were composed of 36 functional genes, including 12 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. All protein-coding genes showed A + T bias, positive GC skews, and negative AT skews in nucleotide composition. Phylogenetic analysis based on the mitochondrial genomes showed that Modiolinae and Bathymodiolinae clustered together to form a sister relationship. Seven Modiolinae species were divided into two clades: L1 (M. modulaides, M. auriculatus and Modiolus philippinarum Hanley, 1843) and L2 [Modiolus modiolus (Linnaeus, 1758), Modiolus kurilensis Bernard, 1983, Modiolus nipponicus (Oyama, 1950), and Modiolus comptus (Sowerby III, 1915)]. The divergence time of the two clades was approximately 105.75 Ma. Furthermore, the transfer RNA gene rearrangement, longer genetic distance, and greater genetic differentiation were confirmed between the L1 and L2 clades, as well as differences in the external characteristics of the shells of the two clades. CONCLUSIONS: Based on the molecular data, it was speculated that species from the L1 clade might belong to other genera or new genera. This study provides molecular information for further taxonomic and phylogenetic studies of Mytilidae.

Mating behaviors in ovoviviparous black rockfish (Sebastes schlegelii): molecular function of prostaglandin E2 as both a hormone and pheromone.

Prostaglandins (PGs) are profound hormones in teleost sexual behavior, especially in mating. PGs act as pheromones that affect the olfactory sensory neurons of males, inducing the initiation of a series of mating behaviors. However, the molecular mechanism by which PGs trigger mating behavior in ovoviviparous teleosts is still unclear. In the present study, we employed the ovoviviparous black rockfish (Sebastes schlegelii), an economically important marine species whose reproductive production is limited by incomplete fertilization, as a model species. The results showed that when the dose of PGE2 was higher than 10 nmol/L, a significant (P < 0.05) increase in mating behaviors was observed. Dual-fluorescence in situ hybridization indicated that PGE2 could fire specific neurons in different brain regions and receptor cells in the olfactory sac. After combining with specific neurons in the central nervous system (CNS), a series of genes related to reproduction are activated. The intracerebroventricular administration of PGE2 significantly increased lhb levels (P < 0.05) in both sexes. Moreover, steroidogenesis in gonads was also affected, inducing an increase (P < 0.05) in E2 levels in males and T levels in females. PGE2 levels were also increased significantly (P < 0.05) in both sexes. The present study revealed that PGE2 can activate mating behavior in black rockfish in both hormone and pheromone pathways, leading to variations in sex steroid levels and activation of reproductive behaviors. Our results provide not only novel insight into the onset of mating behaviors in ovoviviparous teleosts but also solutions for the incomplete fertilization caused by natural mating in cage aquaculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00214-w.

Integrated Metagenomic and Metabolomic Analysis on Two Competing Mussels, Mytella strigata and Perna viridis, in China.

Biological invasion is a primary direct driver of biodiversity loss. Recently, owing to exploitation competition with an invasive mussel, Mytella strigata (Hanley, 1843), there has been a drastic decrease in the population of native Perna viridis (Linnaeus, 1758) in several western Pacific regions. In the present study, intestinal microbiota, metabolome, and key digestive enzyme activities were compared between the two competing mussels, M. strigata and P. viridis, to elucidate the differences in intestinal microbiota and metabolic points. We observed that Proteobacteria, Firmicutes, and Bacteroidota were the three predominant bacterial phyla in the two species. The relative abundance of Bacteroidota related to carbohydrate-degrading ability was significantly higher in M. strigata than in P. viridis. Compared to P. viridis, different metabolites including maltose and trehalose were enriched in M. strigata. Lastly, higher carbohydrases activities of alpha-amylase, cellulase, and xylanase were observed in M. strigata than in P. viridis. These differences might play an important role in the adaptation process of M. strigata to the new environment. This study provides important basic knowledge for investigating the competition between M. strigata and P. viridis in terms of food resources utilization.

PGE2 functions in ovoviviparous teleost black rockfish (Sebastes schlegelii): evolutionary status between parturition and ovulation†.

Fish have evolved various reproductive strategies including oviparity, viviparity, and ovoviviparity, which undoubtedly affect the survival of the whole species continuity. As the final step in reproduction, parturition in viviparous vertebrate and ovulation in oviparous teleost seem to share a similar mechanism, when prostaglandins (PGs) act as the trigger to launch the whole process. In the present study, ovoviviparous teleost black rockfish (Sebastes schlegelii) is employed as the research object. Intraperitoneal injection showed that PGE2 (500 µg/kg) could activate the delivery reactions in perinatal black rockfish. RNA-seq data of ovary in perinatal period revealed transcriptional change in cell junction, inflammation, and apoptosis, which is related to mammal parturition and teleost ovulation. Further results proved the positive correlation between ptger EP2 and previous mentioned pathways. Subsequent experiment proved that PGE2 was able to induce the ovulation and spawning in unfertilized individuals, which had a bilayer follicular structure compared to monolayer follicular in perinatal period black rockfish. Both unfertilized and perinatal ovary matrix could response to PGE2 stimulation. In conclusion, the function of PGE2 in activating both parturition and ovulation in a relatively different pathways conserved with viviparity or oviparity provided novel evidence of the evolutionary status of ovoviviparous vertebrates.

FGFs function in regulating myoblasts differentiation in spotted sea bass (Lateolabrax maculatus).

Fibroblast growth factors (FGFs) are a family of structurally related peptides that regulate processes such as cell proliferation, differentiation, and damage repair. In our previous study, fibroblast growth factor receptor 4 (fgfr4) was detected in the most significant quantitative trait loci (QTL), when identified of QTLs and genetic markers for growth-related traits in spotted sea bass. However, knowledge of the function of fgfr4 is lacking, even the legends to activate the receptor is unknown in fish. To remedy this problem, in the present study, a total of 33 fgfs were identified from the genomic and transcriptomic databases of spotted sea bass, of which 10 were expressed in the myoblasts. According to the expression pattern during myoblasts proliferation and differentiation, fgf6a, fgf6b and fgf18 were selected for further prokaryotic expression and purification. The recombinant proteins FGF6a, FGF6b and FGF18 were found to inhibit myoblast differentiation. Overall, our results provide a theoretical basis for the molecular mechanisms of growth regulation in economic fish such as spotted sea bass.

Genetic Analysis Based on Mitochondrial nad2 Gene Reveals a Recent Population Expansion of the Invasive Mussel, Mytella strigata, in China.

Mytella strigata is a highly adaptable invasive alien species that has been established in coastal China since 2014. Mitochondrial DNA (mtDNA) is an important tool for studying the evolution and population genetics of invasive species. In this study, the mitochondrial genome of M. strigata from China was sequenced by Illumina high-throughput sequencing and characterized with 13 protein-coding genes (PCGs). By assessing the selective pressure of 13 PCGs, the nad2 gene had the fastest evolutionary rate and was finally selected for population genetic analysis. A total of 285 nad2 sequences from seven M. strigata populations in China were analyzed and showed obviously T-rich and C-rich characteristics. According to population genetic diversity analysis, all the seven populations had haplotype (gene) diversity (Hd) ≥ 0.5 and nucleotide diversity (Pi) < 0.005. Haplotype networks showed a "star" distribution. Population historical dynamic analyses showed that Fu's Fs and Tajima's D values of all populations were negative except the Qukou (QK) and Beihai (BH) populations. The Zhangzhou (ZJ) and Xiamen (XM) populations were unimodal while the other populations were multimodal. These results suggested that the population of M. strigata in China may have passed the bottleneck period and is currently in a state of population expansion.

Pro-inflammatory cytokine IL1ß1 participates in promoting parturition related pathways in the ovoviviparous teleost black rockfish (Sebastes schlegelii).

Along with the evolution process, the reproductive strategies evolved including oviparity, viviparity and ovoviviparity, to fit the residential environment maximize the survival rate of the off spring. In mammals, the key to the initiation of parturition is the inflammatory response at the maternal-fetal interface. As a pro-inflammatory cytokine, interleukin 1 beta (IL1ß) plays an important role in the process of human parturition. While less is known about IL1ß1 in teleost parturition, identification of the functions of IL1ß1 in inducing the parturition, black rockfish, an ovoviviparity teleost, which provides over 60% nutrition supply for over 50 000 embryos though a placenta like structure during pregnant, was employed as the research model. In the present study, based on the gene cloning, we detected the expression pattern of both Il1b1 and its receptor perinatal period, as well as the localization to the ovary by in situ hybridization. The different expression genes in transcriptomic data of perinatal primary ovarian cells treated with the recombinant IL1ß1 (rIL1ß1) obtained by prokaryotic expression system were analyzed. Differentially expressed genes, functional enrichment and pathway analysis mainly included immune response, signal transduction and cell death. In summary, our research provides novel insights into the potential role of IL1ß1 in the parturition of ovoviviparity teleost.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors.

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Comparative transcriptomic analysis and genome-wide characterization of the Semaphorin family reveal the potential mechanism of angiogenesis around embryo in ovoviviparous black rockfish (Sebastes schlegelii).

To guarantee the quality and survival rate of their offspring, ovoviviparous teleost evolved special characteristics of in vivo fertilization and embryo development. Maternal black rockfish, having over 50 thousand embryos developing within the ovary simultaneously, provided around 40% nutrition throughout oocyte development, while the capillaries around each embryo contributed the rest 60% during pregnancy. Since fertilization, capillaries started to proliferate and developed into a placenta-like structure that covered over half of each embryo. Aimed to characterize the potential mechanism behind, comparative transcriptome analysis of samples collected according to the process of pregnancy. Three important time point in the process, including mature oocyte stage, fertilization and sarcomere period, were chosen for the transcriptome sequencing. Our study identified key pathways and genes involved in the cell cycle as well as DNA replication and repair, cell migration and adhesion, immune, and metabolic functions. Notably, several of the semaphoring gene family members were differently expressed. To confirm the accuracy of these genes, total of 32 sema genes were identified from the whole genome and distinct expression pattern of sema genes was observed in different pregnant stages. Our results revealed a novel insight for further investigating the functions of sema genes in reproduction physiology and embryo processes in ovoviviparous teleost.

Molecular characterization and expression patterns of glucocorticoid receptors in the viviparous black rockfish Sebastes schlegelii.

Glucocorticoid receptors (GRs) are ligand-activated transcription factors associated with anti-inflammation, stress, metabolism and gonadal development. In this study, two gr genes (gr1 and gr2) were cloned and analyzed from a viviparous teleost, black rockfish (Sebastes schlegelii). The phylogenetic analysis of GRs showed that GR1 and GR2 clustered into teleost GR1 and GR2 separately and differed from the GRs of tetrapods or basal ray-finned fishes. Black rockfish GRs possess four modular domains of the nuclear receptor superfamily: an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR) and a ligand-binding domain (LBD). Nine conserved amino acid inserts were found in the GR1 DBD, and the ligand cavity-related amino acids of GR1 and GR2 LBD were slightly different. Tissue distribution analysis revealed that grs was widely expressed in various tissues, while cyp11b was mainly expressed in the testis and head kidney. The cyp11b transcripts were localized in the interrenal glands of the head kidney, the main source of cortisol; grs transcripts were detected in oocytes, the follicle layer and the ovarian wall. Histologically, significant blood vessel dilation was observed in the fetal membrane during or after parturition of black rockfish. The highest levels of serum cortisol and ovarian cyp11b mRNA were detected in parturition. In addition, the relative expression level of gr1 was upregulated significantly after delivery, while the levels of gr2 showed no significant change. In addition, in vitro GC treatment inhibited the expression of il1b but significantly upregulated the transcription of il1r1. These data provide evidence that GRs are likely to work as anti-inflammatory factors by inhibiting the functions of pro-inflammatory factors in the parturition of black rockfish.