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.

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

The ovarian microenvironment plays a crucial 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 of this process in black rockfish (Sebastes schlegelii) using 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 the early spermatozoa-storage stage (1 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. Before fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promotes spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism that influences 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.

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.

The residue of salinomycin in the muscles of olive flounder (Paralichthys olivaceus) and black rockfish (Sebastes Schlegeli) after oral administration analyzed by LC-Tandem-MS.

BACKGROUND: Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. RESULTS: The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). CONCLUSION: Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

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.

Identification of reactive oxygen species modulator 1 (Romo 1) from black rockfish (Sebastes schlegelii) and deciphering its molecular characteristics, immune responses, oxidative stress modulation, and wound healing properties.

Reactive oxygen species modulator 1 (Romo1) is a mitochondrial inner membrane protein that induces mitochondrial reactive oxygen species (ROS) generation. In this study, we identified the Romo1 homolog from the black rockfish (Sebastes schlegelii), named it as SsRomo1, and characterized it at the molecular as well as functional levels. An open reading frame consisting of 240 bp was identified in the SsRomo1 complementary DNA (cDNA) sequence that encodes a 79 amino acid-long polypeptide with a molecular weight of 8,293 Da and a theoretical isoelectric point (pI) of 9.89. The in silico analysis revealed the characteristic features of SsRomo1, namely the presence of a transmembrane domain and the lack of a signal peptide. Homology analysis revealed that SsRomo1 exhibits the highest sequence identity with its fish counterparts (>93%) and shares a similar percentage of sequence identity with mammals (>92%). Additionally, it is closely clustered together with the fish clade in the constructed phylogenetic tree. The subcellular localization analysis confirmed its mitochondrial localization within the fathead minnow (FHM) cells. Under normal physiological conditions, the SsRomo1 mRNA is highly expressed in the rockfish ovary, followed by the blood and testis, indicating the abundance of mitochondria in these tissues. Furthermore, the significant upregulation of SsRomo1 in cells treated with lipopolysachharide (LPS), polyinosinic:polycytidylic acid, and Streptococcus iniae suggest that the increased ROS production is induced by SsRomo1 to eliminate pathogens during infections. Incidentally, we believe that this study is the first to determine the involvement of SsRomo1 in LPS-mediated nitric oxide (NO) production in RAW267.4 cells, based on their higher NO production as compared to that in the control. Moreover, overexpression of SsRomo1 enhanced the wound healing ability of FHM cells, indicating its high invasion and migration properties. We also determined the hydrogen peroxide-mediated cell viability of SsRomo1-overexpressed FHM cells and observed a significant reduction in viability, which is possibly due to increased ROS production. Collectively, our observations suggest that SsRomo1 plays an important role in oxidative stress modulation upon immune stimulation and in maintenance of tissue homeostasis in black rockfish.

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.

Genome-Wide Identification, Evolutionary Analysis, and Expression Patterns of Cathepsin Superfamily in Black Rockfish (Sebastes schlegelii) following Aeromonas salmonicida Infection.

Cathepsins are lysosomal cysteine proteases belonging to the papain family and play crucial roles in intracellular protein degradation/turnover, hormone maturation, antigen processing, and immune responses. In the present study, 18 cathepsins were systematically identified from the fish S. schlegelii genome. Phylogenetic analysis indicated that cathepsin superfamilies are categorized into eleven major clusters. Synteny and genome organization analysis revealed that whole-genome duplication led to the expansion of S. schlegelii cathepsins. Evolutionary rate analyses indicated that the lowest Ka/Ks ratios were observed in CTSBa (0.13) and CTSBb (0.14), and the highest Ka/Ks ratios were observed in CTSZa (1.97) and CTSZb (1.75). In addition, cathepsins were ubiquitously expressed in all examined tissues, with high expression levels observed in the gill, intestine, head kidney, and spleen. Additionally, most cathepsins were differentially expressed in the head kidney, gill, spleen, and liver following Aeromonas salmonicida infection, and their expression signatures showed tissue-specific and time-dependent patterns. Finally, protein-protein interaction network (PPI) analyses revealed that cathepsins are closely related to a few immune-related genes, such as interleukins, chemokines, and TLR genes. These results are expected to be valuable for comparative immunological studies and provide insights for further functional characterization of cathepsins in fish species.

Effects of Cryopreservation on Sperm with Cryodiluent in Viviparous Black Rockfish (Sebastes schlegelii).

Black rockfish is an economically important fish in East Asia. Little mention has been paid to the sperm cryopreservation in black rockfish. In this study, the optimal cryodiluent was selected from 48 combinations by detecting various sperm parameters. Transcriptome and methylome analysis were further performed to explore the molecular mechanism of inevitable cryoinjuries. The results showed that cryopreservation had negative effects on the viability, DNA integrity, mitochondrial activity, total ATPase and LDH of sperm even with optimal cryodiluent (FBS + 15% Gly). Transcriptome and methylome analysis revealed that the expression of 179 genes and methylation of 1266 genes were affected by cryopreservation. These genes were enriched in GO terms of death, G-protein coupled receptor signaling pathway, response to external stimulus and KEGG pathways of phospholipase D signaling pathway and xenobiotic and carbohydrate metabolism pathways. The role of PIK3CA and CCNA2 were highlighted in the protein-protein interaction network, and the sperm quality-related imprinted gene mest was identified among the 7 overlapping genes between transcriptome and methylome. Overall, the cryodiluent for black rockfish sperm was optimized, providing a feasible method for cryopreservation. The transcriptome and methylome data further demonstrated the underlying molecular mechanisms of cryoinjuries, proving clues for improvement of cryopreservation method of black rockfish.

Expression Analysis of ZPB2a and Its Regulatory Role in Sperm-Binding in Viviparous Teleost Black Rockfish.

Black rockfish is a viviparous teleost whose sperm could be stored in the female ovary for five months. We previously proposed that zona pellucida (ZP) proteins of black rockfish play a similar sperm-binding role as in mammals. In this study, SsZPB2a and SsZPB2c were identified as the most similar genes with human ZPA, ZPB1 and ZPB2 by Blastp method. Immunohistochemistry showed that ovary-specific SsZPB2a was initially expressed in the cytoplasm of oocytes at stage III. Then it gradually transferred to the region close to the cell membrane and zona pellucida of oocytes at stage IV. The most obvious protein signal was observed at the zona pellucida region of oocytes at stage V. Furthermore, we found that the recombinant prokaryotic proteins rSsZPB2a and rSsZPB2c could bind with the posterior end of sperm head and rSsZPB2a was able to facilitate the sperm survival in vitro. After knocking down Sszpb2a in ovarian tissues cultivated in vitro, the expressions of sperm-specific genes were down-regulated (p < 0.05). These results illustrated the regulatory role of ZP protein to the sperm in viviparous teleost for the first time, which could advance our understanding about the biological function of ZP proteins in the teleost.

Comparative transcriptomic analysis of gonadal development and renewal in the ovoviviparous black rockfish (Sebastes schlegelii).

BACKGROUND: The black rockfish (Sebastes schlegelii) has an ovoviviparous reproductive pattern and long-term sperm storage, resulting in asynchronous gonadal development between the sexes. However, the comprehensive understanding of gonadal development in black rockfish has not yet been achieved. Here, we studied gonadal development and germ cell renewal using histology and RNA-seq. RESULTS: In this study, RNA-seq was performed on testes and ovaries to characterize key pathways and genes that are active during development and gamete maturation in black rockfish. Differentially expressed genes (DEGs) were identified and annotated in 4 comparisons (F_III vs. F_IV, F_IV vs. F_V, M_III vs. M_IV and M_IV vs. M_V). Based on analysis of DEGs enriched in the testis, 11 and 14 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mapped to the M_III vs. M_IV group and the M_IV vs. M_V group, respectively. DEGs in ovarian development were also classified into 10 groups according to their biological functions. The expression patterns of the selected genes determined by qPCR were significantly correlated with the RNA-Seq results, supporting the reliability and accuracy of the RNA-Seq analysis. E2 levels showed down regulation from previtellogenesis to mature stage in female and T level showed down regulation from spermatogenesis to regressed stage in the male. CONCLUSIONS: The categories "intercellular interaction and cytoskeleton", "molecule amplification" and "repair in the cell cycle" were revealed to be crucial in testis development and spermatogenesis, as was the biosynthesis of a series of metabolites. Our results provide comprehensive insight into black rockfish gonadal development and provide a basis for further study of reproductive physiology and molecular biology in ovoviviparity teleosts.

Identification of tissue-specific expression of CXCL14 in black rockfish (Sebastes schlegelii).

CXCL14 is a chemokine which is orthologous in mammals and fish. CXCL14 has a functional role in different organs, with immunomodulatory functions in mammals, but its expression and function in fish is not well known. Moreover, it shows no effects related to immunity in the central nervous system or the reproductive tract in diverse species. Black rockfish (Sebastes schlegelii) is an economically important fish in Asian countries, whose CXCL14 expression pattern is yet to be understood. In this study, the homology of the CXCL14 amino acid sequence in S. schlegelii was compared with that in other species, including fish. Moreover, in situ hybridization analysis revealed that it was highly expressed in the brain and ovary of S. schlegelii. Taken together, we identified for the first time, the cell-specific expression of CXCL14 in S. schlegelii.

Characterization of CYP11A1 and its potential role in sex asynchronous gonadal development of viviparous black rockfish Sebastes schlegelii (Sebastidae).

Mitochondrial cytochrome P450 side-chain cleavage (P450scc), encoded by the cyp11a1 gene, initiates the first step of steroid biosynthesis. In this study, a 1554-bp open reading frame (ORF) of black rockfish (Sebastes schlegelii) cyp11a1 was cloned. The cyp11a1 gene is located on chromosome 5 and has 9 exons. The ORF encodes a putative precursor protein of 517 amino acids, and the predicted cleavable mitochondrial targeting peptide is located at amino acids 1-39. P450scc shares homology with other teleosts and tetrapods, which have relatively conserved binding regions with heme, cholesterol and adrenodoxin. Tissue distribution analysis revealed that the highest expression levels of cyp11a1 were detected in mature gonads and head kidney but that low levels were detected in gestational/regressed ovaries, regressed testes and other tissues. Immunostaining of P450scc was observed in testicular Leydig cells, ovarian theca cells, interrenal glands of head kidney, pituitary and multiple regions of brain. Particularly, two kinds of fish-specific P450scc-positive cells, including coronet cells of brain saccus vasculosus and hypophyseal somatolactin cells, were identified in black rockfish. Our results provide novel evidence for the potential role played by P450scc in reproduction behavior by mediating steroidogenesis in viviparous teleost.

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii).

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11ß-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17ß-estradiol (E2). The results showed cyp11a was significantly increased at 10-6  mol ml-1 of 17α-MT and 10-8  mol ml-1 of E2 in ovary and 10-10  mol ml-1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.

Gonadal lipidomics profile of an ovoviviparity teleost, black rockfish, during gonadal development.

In order to study the variation of gonad lipidomics during reproductive cycle, black rockfish was employed as the research model in the present study. Using histology, lipidomics, and qPCR, the profile of gonad lipidomics and the expression levels of related genes during different developmental stages were detected and analyzed to show the potential regulatory network of lipid metabolism. Based on Ultra High-Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometry (UHPLC-QTOFMS), four significant differential glycerophospholipid metabolic pathways including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA) were enriched by KEGG. Pathway-related enzyme-coding genes, including phosphatidylserine decarboxylase (pisd), phosphatidylserine synthase (ptdss1, ptdss2), and phospholipase D (pld1, pld2) were identified from the whole genome data and confirmed by cloning. The expression profiles of these genes were tested by qPCR in the tissues and gonads in developmental stages, and we found that pisd, pld, and ptdss genes were all downregulated through the developmental process in the brain of male, and the latter two genes were upregulated in the liver and testis at stage IV, which were the opposite trend observed in the female. Thus, our findings would be helpful in further understanding the substance metabolism and regulation during gonad development in ovoviviparity teleosts.

Altered physiological response and gill histology in black rockfish, Sebastes schlegelii, during progressive hypoxia and reoxygenation.

Hypoxia has gradually become common in aquatic ecosystems and imposes a significant challenge for fish farming. The loss of equilibrium (LOE), 50% lethal time (LT50), plasma cortisol, glucose, red blood cells (RBC), hemoglobin (Hb), gill histological alteration, and related parameters (lamellar length [SLL] and width [SLW], interlamellar distance [ID], basal epithelial thickness [BET], lamellar surface area [LA], and gill surface area [GSA]); respiratory rate; the proportion of the secondary lamellae available for gas exchange (PAGE); and hypoxia-inducible factor (hif-1α, hif-2α) mRNA expression were determined during progressive hypoxia and reoxygenation (R-0, R-12, R-24 h) to illustrate the underlying physiological response mechanisms in black rockfish Sebastes schlegelii. Results showed that the DO concentration significantly decreased during progressive hypoxia, while DO at LOE and LT50 were 2.42 ± 0.10 mg L-1 and 1.67 ± 0.38 mg L-1, respectively. Cortisol and glucose were significantly increased at LOE and LT50, with the highest levels observed at LT50, and then gradually recovered to normal within reoxygenation 24 h. RBC number and Hb results were like those of glucose. Hypoxia stress resulted in lamellar clubbing, hypertrophy, and hyperplasia. Respiratory frequency significantly increased at LOE and decreased at LT50. Lamellar perimeters, SLL, ID, LA, GSA, and PAGE, significantly increased at LOE and LT50, with the highest values observed at LT50. However, SLW and BET significantly decreased at LOE, LT50, and R-0. These parameters recovered to nearly normal levels at R-24 h. hif-1α mRNAs in gill and liver were significantly upregulated at LOE and LT50, and recovery to normal after reoxygenation 24 h. hif-2α mRNAs in gill was similar to that of hif-1α, whereas hepatic hif-2α mRNAs remained unchanged during hypoxia-reoxygenation. These results indicated that progressive hypoxia stress elevated RBC number, Hb, cortisol, and glucose levels, induced the alteration of gill morphology, increased LA and GSA, stimulated respiratory frequency and PAGE, and upregulated the transcription of hif-1α and hif-2α in gill and liver. Reoxygenation treatment for 24 h alleviated the stress mentioned above effects. These findings expand current knowledge on hypoxia tolerance in black rockfish Sebastes schlegelii.

Full length transcriptome profiling reveals novel immune-related genes in black rockfish (Sebastes schlegelii).

Lacking full-length transcriptome for black rockfish (Sebastes schlegelii) limits novel gene discoveries and gene structures analysis. Therefore, we constructed the full-length transcriptome of black rockfish using Single-Molecule Real-Time Sequencing technology. Totally, we produced 21.73 Gb raw reads containing 298,904 circular consensus sequence (CCS) reads. Full-length (FL) and Non-full-length (NFL) isoforms were obtained based on the presence of 5' and 3' primers as well as poly (A) tails. The results showed 70.71% reads were identified as FL isoforms. Moreover, the average length of these PacBio isoforms is 2,632 bp, which is much longer than the length of the unigenes with the average length of 589 bp which generated from Illumina platform. Meanwhile, we identified 43,068 non-redundant transcripts, 12,485 alternative splicing (AS), 6,320 polyadenylation (APA) and 499 gene fusions as well as numerous long non-coding RNAs based on mapped FL isoforms. In addition, we identified 147 and 528 immune-related genes from novel genes and unmapped transcripts. The provided dataset can be utilized to discover novel genes and construct a comprehensive transcript dataset for black rockfish.

Waterborne manganese modulates immunity, biochemical, and antioxidant parameters in the blood of red seabream and black rockfish.

Immunotoxic effects of manganese (Mn) were investigated in the blood of the economically important marine fish, red seabream (Pagrus major) and black rockfish (Sebastes schlegelii) when exposed to different concentrations of Mn (0, 0.5, 1, and 2 mg L-1) for 14 days. During exposure, the levels of alternative complement activity in both fish were significantly lowered at 2 mg L-1 of Mn of exposure. Lysozyme activity was significantly decreased in black rockfish in all concentrations of Mn after 14 days, while in red seabream, the decrease was significant with concentrations of 1 and 2 mg L-1 of Mn after 7 and 14 days of exposure. A significantly low level was observed only in the 2 mg L-1-exposed red seabream on day 14 of exposure. The concentrations of hemoglobin, red blood cells, white blood cells, and total serum proteins were significantly decreased in both fish under exposure to 1 and 2 mg L-1 of Mn, while cortisol, alanine transferase, aspartate transaminase, and alkaline phosphatase levels were significantly increased compared to the levels of control groups. No significant change was found in serum glucose and albumin except in red seabream exposed to 2 mg L-1 of Mn for 14 days. The responses of the antioxidant defense system were significantly induced in both fish after exposure to 1 and 2 mg L-1 of Mn on day 7 and 14 of exposure. Taken together, alterations of these parameters suggest the immunotoxicity of waterborne Mn produced by the modulation of hematological components and the induction of oxidative stress in the blood of these marine fish.

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, Sebastes schlegelii.

C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from Sebastes schlegelii was identified and designated as SsC1inh. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, SsC1inh mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish.

A teleostan homolog of catalase from black rockfish (Sebastes schlegelii): insights into functional roles in host antioxidant defense and expressional responses to septic conditions.

Antioxidative defense renders a significant protection against environmental stress in organisms and maintains the correct redox balance in cells, thereby supporting proper immune function. Catalase is an indispensable antioxidant in organisms that detoxifies hydrogen peroxides produced in cellular environments. In this study, we sought to molecularly characterize a homolog of catalase (RfCat), identified from black rockfish (Sebastes schlegelii). RfCat consists of a 1581 bp coding region for a protein of 527 amino acids, with a predicted molecular weight of 60 kD. The protein sequence of RfCat harbored similar domain architecture to known catalases, containing a proximal active site signature and proximal heme ligand signature, and further sharing prominent homology with its teleostan counterparts. As affirmed by multiple sequence alignments, most of the functionally important residues were well conserved in RfCat. Furthermore, our phylogenetic analysis indicates its common vertebrate ancestral origin and a close evolutionary relationship with teleostan catalases. Recombinantly expressed RfCat demonstrated prominent peroxidase activity that varied with different substrate and protein concentrations, and protected against DNA damage. RfCat mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, RfCat mRNA expression was modulated in response to pathogenic stress elicited by Streptococcus iniae and poly I:C in blood and spleen tissues. Collectively, our findings indicate that RfCat may play an indispensable role in host response to oxidative stress and maintain a correct redox balance after a pathogen invasion.