Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C₁₈Δ6/C₂₀Δ5 but not C₂₂ PUFA substrates.     

Complete mitochondrial genome of black porgy Acanthopagrus schlegelii (Perciformes, Sparidae)

Black porgy, Acanthopagrus schlegelii, is a marine protandrous hermaphrodite and belongs to one of the most important species commercialized for food in various areas of Asia. In this study, the complete mitochondrial genome of A. schlegelii has been determined. The mitogenome was 16,649 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 non-coding regions. It shared 90.2%, 82.3%, and 82.1% mitogenome sequence with Acanthopagrus latus, Parargyrops edita, and Pagrus major, respectively.     

Age and growth of black seabream Acanthopagrus schlegelii (Sparidae) in Hong Kong and adjacent waters of the northern South China Sea

Age and growth of the black seabream Acanthopagrus schlegelii (family Sparidae) from the northern South China Sea (NSCS) were studied by reading growth rings in sectioned sagittal otoliths. Ring formation frequency was determined to be annual by using marginal increment analysis. The von Bertalanffy growth function parameters were estimated as: L_∞ = 43.7 cm L_S; K =0.22 year; t₀ = ?1.59 years. Functional males are significantly younger than functional females, with sexually transitional individuals between the modal ages of males and females supporting protandry in this species. Males become sexually mature within 1 year and 50% age at sex change is at 2 years. The maximum age recorded for both males and females sampled was 9 years which is lower than for conspecifics elsewhere and may reflect high fishing pressure in the study area when compared with conspecifics in other areas or could reflect latitudinal effects. Otolith mass was significantly and positively related to age, providing a cheap and quick alternative method for approximating age. Acanthopagrus schlegelii is a relatively fast‐growing and rapidly maturing species attaining a similar asymptotic length to conspecifics. A need for fishery management is indicated to protect both young juveniles and older adults, especially females, to increase reproductive output and safeguard fishery production.     

Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

Molecular characterization, expression, and functional analysis of two thioredoxins in the black rockfish (Sebastes schlegelii)

Thioredoxins (TRxs) are a family of small evolutionarily conserved proteins that are essential for the maintenance of cellular homeostasis. Two TRx homologue cDNAs were isolated from a black rockfish concanavalin A (Con A)/phorbol myristate acetate (PMA)-stimulated leucocyte cDNA library and named BrTPx1-1 and BrTPx1-2. As compared with other known TRx peptide sequences, the most conserved regions of both BrTRx1-1 and BrTRx1-2 peptides were found to be the redox-active site Trp-Cys-X-X-Cys (WCXXC). The TRx present in most species is a TRx1-2 protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in the larger 13 kDa BrTRx1-1 protein, a Cys-Pro-Pro-Cys (CPPC) active site was identified. Here, we report the identification of a new member of the TRx protein family from the teleost black rockfish, which defines a new subclass of 13-kDa TRx1-1 proteins. Phylogenetic analysis indicated that both BrTRx1-1 and BrTRx1-2 were grouped with other vertebrate TRx1 peptides. BrTRx1-1 expression was strongly induced in peripheral blood leucocytes (PBLs) 12-24 h following Con A/PMA stimulation, with peak expression at 24 h post-stimulation. BrTRx1-2 was induced in PBLs after stimulation with lipopolysaccharide (LPS), Con A/PMA, or poly I:C at 24 h. The BrTRx1-1 gene was predominantly expressed in the liver and gills, while BrTRx1-2 was expressed in PBLs and gills. After treatment with a high concentration (10 μg/mL) of rBrTRx1-1 or rBrTRx1-2, kidney leucocytes exhibited increased cell proliferation and viability under oxidative stress.     

Molecular characterization of hepcidin AS-hepc2 and AS-hepc6 in black porgy (Acanthopagrus schlegelii): expression pattern responded to bacterial challenge and in vitro antimicrobial activity

There are more diversified isoforms of the hepcidin gene that exist in fishes than in mammals, and elucidating the differences between these isoforms should provide insight into the functioning of hepcidin in fishes. In our study, AS-hepc2 and AS-hepc6 hepcidin isoforms from black porgy were characterized for their in vivo expression patterns following bacterial challenge, and their in vitro antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. As a result, two isoforms were observed to be widely distributed in all the tissues tested. AS-hepc2 was a liver-expressed hepcidin peptide which was always highly more expressed in the liver than in the other tissues tested no matter whether this was before or after bacterial challenge. AS-hepc6 was detected mainly in the head kidney and trunk kidney of normal fish, but, in the challenged fish, its expression involved more tissues than just the kidneys. The mature peptides of AS-hepc2 and AS-hepc6 were modeled for 3D structure and then synthesized for antimicrobial assay. AS-hepc6 had a wider antimicrobial spectrum than AS-hepc2 and, in particular, had more potent antifungal activity. Our study indicated that the two hepcidin isoforms had different characteristics in terms of their expression patterns and antimicrobial activity, and they were assumed to play an overlapping role in the innate immune system of black porgy against invading pathogens.     

Reproductive biology of black seabream Acanthopagrus schlegelii,threadfin porgy Evynnis cardinalis and red pargo Pagrus major in the northern South China Sea with consideration of fishery status and management needs

The reproductive biology of three commercially significant seabream species (family: Sparidae) Acanthopagrus schlegelii, Evynnis cardinalis and Pagrus major, taken from Hong Kong and adjacent northern South China Sea (SCS) waters, were investigated for their sexual patterns, spawning seasons, length at maturity and exploitation in relation to their conservation and management status. Histological analysis showed E. cardinalis and P. major to be functionally gonochoristic, the latter having a bisexual juvenile stage and being a rudimentary hermaphrodite. Acanthopagrus schlegelii is a protandric hermaphrodite. Standard length (L_S) at 50% sex change for A. schlegelii is 291 mm. L_S at 50% female maturity for E. cardinalis and P. major are 117 and 332 mm, respectively. For all three species, the spawning period falls between November and March. The study highlights geographical differences in reproductive biology among the species and a paucity of fishery or other population‐related data. While heavy fishing pressure, life‐history characteristics and absence of effective management throughout the geographic ranges of these species make them susceptible to overfishing, they nonetheless appear to be generally more resilient than many other taxa that comprise the multi‐species fisheries of the region, possibly due to their relatively rapid sexual maturation and spatial movement patterns. Overall, however, little is known of the biology, fishing history and current fishery status of sparids in general in the northern SCS and the current study is one of the first to examine such aspects of this family in the region and to consider appropriate management options.     

The identification of fish eggs of two species,the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii

Fish eggs of the ovate sole Solea ovata and black porgy Acanthopagrus schlegelii were identified through DNA barcoding of cytochrome c oxidase subunit I (COX1). Visual taxonomic features were achieved, and photographs of the eggs of both species at different developmental stages were reported for the first time. In addition, the dissolution of oil globules caused by ethanol as egg fixatives was observed. This result showed the importance of using formalin as egg fixatives in the case of morphometric analysis and the necessity of combining molecular and visual taxonomic method for morphological study.     

Fine-scale genetic structure derived from stocking black sea bream, Acanthopagrus schlegelii (Bleeker, 1854), in Hiroshima Bay, Japan

Black sea bream ( Acanthopagrus schlegelii ) is an important commercial and sport fishing species inhabiting Hiroshima Bay, where an intensive stock enhancement program is carried out for this species. In order to clarify the fine-scale genetic effects of the releases, black sea bream specimens were collected at five locations (Ninoshima, Atatajima, Miyajima, Oonasamijima and Kurahashi) in Hiroshima Bay. High homogeneity was observed among locations. The sample from Ninoshima, where stocking was most intense, presented the lowest number of alleles per locus (13.5) and showed significant differences in the pairwise F _ST value compared to the fish at Atatajima, Miyajima and Oonasamijima, but not significantly different from those collected at Kurahashi. However, all differences disappeared once analysis was performed standardizing the age-classes of all samples. The results suggest an important effect of the releases on genetic diversity of A. schlegelii in Hiroshima Bay. Moreover, the observed genetic population substructure is presumed to be related to the year-class composition of the samples at each location.     

Resolution of the Acanthopagrus black seabream complex based on mitochondrial and amplified fragment-length polymorphism analyses

In this study, DNA analyses were employed to verify the identity of six morphologically similar species that occur in the coastal waters of Taiwan: the black seabream complex (Acanthopagrus latus, Acanthopagrus schlegelii, Acanthopagrus sivicolus, Acanthopagrus taiwanensis, Acanthopagrus chinshira and Acanthopagrus pacificus). Amplified fragment-length polymorphism (AFLP) analyses clearly distinguished the same six species that are morphologically diagnosable based on subtle differences in scale counts and anal-fin colouration. In contrast, mitochondrial DNA analyses based on cytochrome b gene sequences did not distinguish individuals of A. schlegelii and A. sivicolus, reflecting either historical introgression or recent speciation and incomplete sorting of their mitochondrial lineages. Phylogenetic relationships among these six north-west Pacific Ocean species of Acanthopagrus analysed using AFLP data were consistent with scale rows above the lateral line (TRac), sperm ultrastructure and geographical distribution. The study provides molecular tools for future research relevant to improved management of these resources, and an increased understanding of the evolutionary history of this radiation.     

Genetic evidence for the mating system and reproductive success of black sea bream (Acanthopagrus schlegelii)

Understanding the mating system and reproductive success of a species provides evidence for sexual selection. We examined the mating system and the reproductive success of captive adult black sea bream (Acanthopagrus schlegelii), using parentage assignment based on two microsatellites multiplex PCR systems, with 91.5% accuracy in a mixed family (29 sires, 25 dams, and 200 offspring). Based on the parentage result, we found that 93.1% of males and 100% of females participated in reproduction. A total of 79% of males and 92% of females mated with multiple partners (only 1 sire and 1 dam were monogamous), indicating that polygynandry best described the genetic mating system of black sea bream. For males, maximizing the reproductive success by multiple mating was accorded with the sexual selection theory while the material benefits hypothesis may contribute to explain the multiple mating for females. For both sexes, there was a significant correlation between mating success and reproductive success and the variance in reproductive success of males was higher than females. Variation in mating success is the greatest determinant to variation in reproductive success when the relationship is strongly positive. The opportunity for sexual selection of males was twice that of females, as well as the higher slope of the Bateman curve in males suggested that the intensity of intrasexual selection of males was higher than females. Thus, male–male competition would lead to the greater variation of mating success for males, which caused greater variation in reproductive success in males. The effective population number of breeders (N_b) was 33, and the N_b/N ratio was 0.61, slightly higher than the general ratio in polygynandrous fish populations which possibly because most individuals mated and had offspring with a low variance. The relatively high N_b contributes to the maintenance of genetic diversity in farmed black sea bream populations.     

Isolation and characterization of eight microsatellite loci from Picnic seabream (Acanthopagrus berda)

Acanthopagrus berda (Sparidae) is abundant in estuaries and inshore waters of the tropical Indo‐Pacific oceans. Because of the drastic decrease in the species’ wild populations, there is an urgent need to understand its population structure. Eight microsatellite loci were isolated from A. berda. An analysis of 50 individuals from Tapeng Lagoon, southwestern Taiwan demonstrated a low degree of diversity. The allelic numbers ranged from nine to 20. The observed heterozygosity (H_O) ranged from 0.28 to 0.64 (mean, 0.45). We also investigated the prospective value of the microsatellite primers to examine whether they were applicable to related taxa.     

Molecular characterization and expression of metallothionein from freshwater pearl mussel,Hyriopsis schlegelii

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys–Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.     

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 11β-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 17β-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.     

Significant population genetic structure of yellowfin seabream Acanthopagrus latus in China

The yellowfin seabream Acanthopagrus latus is widely distributed throughout the Indo‐West Pacific. The genetic analyses of mtDNA control region sequence variation in samples from Chinese waters revealed a pattern of genetic structure between southern and northern samples (P < 0·05) and high levels of genetic diversity in this species. Significant isolation by distance between southern and northern locations (Mantel test r = 0·35, P = 0·04) and among northern populations (r = 0·55, P = 0·01) indicated that ocean straits and other barriers substantially limit gene flow. Phylogeographical analysis also revealed two major divergent mtDNA lineages across Chinese waters. For conservation, A. latus populations in Chinese waters should be divided into at least two management units for protection.     

Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli

The effects of food deprivation on the hepatic level growth hormone receptor (GHR) were investigated in black seabream (Acanthopagrus schlegeli) both at the protein level (by radioreceptor assay) and at the mRNA level (by ribonuclease protection assay). Serum levels of growth hormone (GH) and triiodothyronine (T₃) were also measured. Condition factor and hepatic proximate composition of the fish were also assessed. Significant decrease in hepatic GHR binding was recorded as early as on day 2 of starvation. On day 30 this decrease was even more pronounced, with the level in the starved fish reaching less than 20% the fed control level. A concomitant decrease in the hepatic GHR mRNA content was also noted during this period, with a progressive decrease from day 2 to day 30 of starvation. The extent of decrease in the mRNA content was less pronounced than the decrease in receptor binding, with the hepatic GHR mRNA content in the day 30 starved fish representing approximately 30% of the level in the fed control. In large contrast, serum GH level increased progressively during starvation. After 30 days of starvation, serum GH levels in the starved fish were more than three times the concentration found in the fed control. Serum T₃ levels, on the other hand, decreased during starvation, with the difference reaching significance on day 15 and day 30. After 30 days of starvation, serum T₃ levels in the starved fish were only approximately 40% the concentration found in the fed control. The hepatic lipid content exhibited an increasing trend during starvation. On day 30 the hepatic lipid content of the starved fish had doubled the level found in the fed control. However, the hepatic protein content did not exhibit much change during starvation. There was also a minor decrease in the moisture content of the liver during starvation, but the condition factor of the fish as a whole registered a gradual decrease during the course of food deprivation.     

Oocyte maturation-inducing steroids in protandrous black porgy, Acanthopagrus schlegeli

The study objectives aimed to investigate the maturation-inducing steroid (MIS) in marine protandrous black porgy, Acanthopagrus schlegeli. The characteristics of oocyte maturation were also described. Females were injected with two successive doses of LHRH analog (LHRH-A, 10 and 50 microg/kg of fish). The ovarian tissue was obtained at 6-h intervals for in vitro oocyte maturation. Both 17,20 beta-dihydroxy-4-pregnen-3-one (DHP) and 17,20 beta,21-trihydorxy-4-pregnen-3-one (20 beta-S) were the most effective steroids to induce in vitro maturation (e.g. germinal vesicle breakdown, GVBD) in oocytes cultured for either 24 h or 1 min. 20 beta-S had a better potency than DHP in inducing oocyte maturation. 17-hydroxyprogesterone, 11-deoxycortisol, and 20 beta-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation at high concentrations. The process of oocyte maturation (after the injection of LHRH analog) was founded to be divided into four stages: hormone-insensitive stage (insensitive to gonadotropin and MIS); MIS-insensitive (respond to gonadotropin, but not MIS); MIS-sensitive (respond to MIS); and spontaneous stage (GVBD in the hormone-free condition), respectively. Cycloheximide blocked GVBD at the MIS-insensitive stage, control (hormone-free), and hormone-induced GVBD at the MIS-sensitive stage in a dose-dependent effect.