Macroscopic and microcosmic observation of the ovary at different stages of reproductive process. a Ovary at the stage of post-mating. Its overall color is yellow, because of fewer blood vessels in the ovary. b Ovary after fertilization, at the early stage of gestation. Its overall color is pink or red, because the well-developed blood vessels spread over the surface of egg membrane. c Ovary at the final stage of gestation. Its overall color is dark, because of developed pigment. The silvery reflections are the individual eyes of the larvae within the ovary. As time goes by, the ovary will enlarge the volume and the ovarian wall will become very thin until the finish of parturition. d Ovary post parturition. Most embryos came out from cloacal orifice via abdomen extrusion. e Ovary before mating. Oocytes are opaque. Only a few of blood vessels were observed. f Ovary after mating. Oocytes develop further, becoming transparent. g Ovary before fertilization. Oocytes are fully developed and ready for fertilization. The highly vascular membranous tissue adjacent to the egg membrane. h Ovary during gestation. The eyes of embryo formed

Schematic diagram of gene location and protein structure. a Schematic diagram of gene location. b Schematic diagram of conserved domains. Domain analysis was performed by SMART online tool base on amino acid sequence. TM transmembrane domain, shown in purple, LC low complexity domain, shown in orange; other structural region, shown in green. c Schematic diagram of 3D structure. Structure prediction analysis was performed via Phyre2 online tool and

modified by PyMol software. Models in line a were shown as cartoon style. Models in line a’ were shown as dot style

Phylogenetic analysis of Bdkrb2 paralogs and orthologs in vertebrates. The phylogenetic tree was constructed by MrBayes software. The length of the branch represents genetic distance; posterior probabilities are shown as numbers on the branches; mcmc = 5,000,000; tree was rooted with the sequence of Callorhinchus milii. Cmi, Callorhinchus milii; Dre, Danio Rerio; Lcr, Larimichthys crocea; Loc, Lepisosteus oculatus; Ola, Oryzias latipes; Pfo, Poecilia Formosa; Xma, Xiphophorus maculatus; Ssc, Sebastes schlegelii

Tissue expression pattern of Bdkrb2 genes. a Heatmap was constructed by comparing different tissues. The x‐axis showed sampled tissues, with the prefix F_ for female and M_ for male samples, and the y‐axis shows genes. The color scale showed standardized TPM values normalized by Z-score method. Gene expression patterns could be roughly divided into gill-type, intestine-type and reproductive organ-type. b The expressions of Bdkrb2 genes in ovarian wall at the stage of post-mating and pre-fertilization. The x‐axis showed genes and the y‐axis shows TPM (Transcripts Per Million). The numeric data were presented as mean ± SD. Statistical significance was tested by one-way ANOVA followed by Tukey’s HSD (harmonic mean sample size = 3, degrees of freedom = 7, subset for alpha = 0.05) using SPSS 20.0 (SPSS, Armonk, NY, USA) and accepted when p < 0.05

Functional study of bdkrb2 genes in zebrafish. Overexpression of Ssc_10023113 and Ssc_10023117 affects the phenotype and vascular development of embryos. An amount of 1.5 ng/embryo of mRNA was injected into Transgenic stain (Fli1a: EGFP) embryos at 1–4 cell stages. All embryos are shown with anterior to the left. a Phenotype of embryos with overexpression of mRNA at 48 h post-fertilization (hpf). Scale bar = 200 μm. a control group, embryos without injection. f control group, embryos with injection of equal volume of sterilized DEPC water. B–e, experimental group, embryos with injection of Ssc_10023113 mRNA. b normal phenotype; c mild phenotype; d medium phenotype; d serious phenotype. g–j, experimental group, embryos with injection of Ssc_10023117 mRNA. g normal phenotype; h mild phenotype; i medium phenotype; j serious phenotype. b Deformation percentages of embryos in each category as shown in (a). Phenotypes were divided into four categories: normal, shown in blue; mild, shown in purple; medium, shown in yellow; serious, shown in pink. N is the number of total samples analyzed in each group. c Vascular abnormalities of embryos with overexpression of mRNA at about 55 hpf. Scale bar = 100 μm. a’, control group, embryos with injection of equal volume of sterilized DEPC water. b’-d’, experimental group, embryos with injection of Ssc_10023113 mRNA. e’–g’, experimental group, embryos with injection of Ssc_10023117 mRNA. Yellow arrowheads indicate different types of vessels, whereas the red asterisks point out the abnormalities in the experimental group. H heart, CCV common cardinal veins, Se intersegmental vessel

Expression pattern of Bdkrb2 genes in the ovary in the reproductive cycle. a Heatmap was constructed by comparing three kinds of samples at different stage of the reproductive cycle. The x‐axis shows sampled tissues with the prefix C. for connective tissue rich in blood vessels covering the egg membrane and E. for embryos and O. for ovarian wall. Arabic numerals represent different stages. 1, pre-fertilization; 2, 1-cell; 3, 8-cells; 4, 16-cells; 5, gastrula stage; 6, 8-somites stage; 7, tailbud stage; 8, pre-hatching; 9, hatching. The color scale shows standardized TPM values normalized by Z-score method