Junbin Zhang

Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus.

In the present study, we cloned the full-length cDNAs of FOXL2, Cyp19a1a and Cyp19a1b and analyzed their expression patterns during gonadal development in spotted scat, Scatophagus argus. All three genes were expressed in ovaries and testes but showed sexual dimorphism. At early stages of gonadal development, the expression of FOXL2 in ovaries was higher than testes. FOXL2 expression deceased gradually as gonadal development continued, and reached the lowest level at the mature stage. Cyp19a1a and Cyp19a1b were expressed coordinately with FOXL2, except at the early vitellogenic stage in the ovary. The expression of FOXL2, Cyp19a1a and Cyp19a1b was mainly localized in granulosa cells of ovaries. In S. argus testes, strong expression of FOXL2 gene was observed in the interstitial cells including tubules and Leydig cells, while Cyp19a1a and Cyp19a1b were mainly expressed in Sertoli cells throughout gametogenesis. These results show that FOXL2 plays an essential role in sexual development, and imply that it may regulate Cyp19a1a and Cyp19a1b expression in S. argus.

Identification of fxyd genes from the spotted scat (Scatophagus argus): molecular cloning, tissue-specific expression, and response to acute hyposaline stress.

By interacting with Na(+), K(+)-ATPase (NKA), the FXYD domain-containing ion transport regulator (FXYD) is involved in teleost osmoregulation, but knowledge of FXYD in marine fish is limited. In the present study, fxyd11 and fxyd12 were identified from the spotted scat (Scatophagus argus), and the two members of the FXYD protein family were expressed in a tissue-specific manner. Fxyd11 mRNA was predominantly expressed in gills, whereas fxyd12 mRNA was mainly distributed in kidneys and intestines. Acute hyposaline stress altered the activity of NKA and the expression of fxyd11 and fxyd12 in gills, kidneys, and intestines. Branchial fxyd11 mRNA expression remained at a low level during freshwater acclimation, whereas NKA activity increased, showing a negative correlation that differed from previous reports. Similarly, renal expression of fxyd11 and fxyd12 mRNA was negatively correlated with NKA activity. Unlike in gills and kidneys, intestinal NKA activity and mRNA expression of fxyd11 and fxyd12 were comparably suppressed. Taken together, the salinity-dependent expression of fxyd11 and fxyd12, and correlation with NKA activity suggested that both fxyd11 and fxyd12 were involved in the response to acute hyposaline challenge in the spotted scat.

Comparative renal gene expression in response to abrupt hypoosmotic shock in spotted scat (Scatophagus argus)

Scatophagus argus, a euryhaline fish, is notable for its ability to tolerate a wide range of environmental salinities and especially for its tolerance to a rapid, marked reduction in salinity. Therefore, S. argus is a good model for studying the molecular mechanisms mediating abrupt hyperosmoregulation. The serum osmotic pressure decreased steeply within one hour after transferring S. argus from seawater (SW) to freshwater (FW) and remained at new balance throughout the duration of one week. To explain this phenomenon and understand the molecular responses to an abrupt hypoosmotic shock, hypoosmotic stress responsive genes were identified by constructing two suppression subtractive hybridization (SSH) cDNA libraries from the kidneys of S. argus that had been transferred from SW to FW. After trimming and blasting, 52 ESTs were picked out from the subtractive library. Among them, 11 genes were significantly up-regulated (p < 0.05). The kinetics studies of gene expression levels were conducted for 1 week after the transfer using quantitative real-time PCR. A significant variation in the expression of these genes occurred within 12h after the hypoosmotic shock, except for growth hormone (GH) and polyadenylate binding protein 1 (PBP1), which were significantly up-regulated 2 days post-transfer. Our results suggest different functional roles for these genes in response to hypoosmotic stress during the stress response phase (1 hpt-12 hpt) and stable phase (12 hpt-7 dpt). Furthermore, the plasma growth hormone level was detected to be significantly elevated at 1 hpt and 24 hpt following abrupt hypoosmotic shock. Meanwhile, several hematological parameters, hemoglobin (HGB), red blood cell (RBC) and mean cellular hemoglobin concentration (MCHC), were observed to be significantly increased at 12 hpt and 2 dpt compared with that of control group. Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.

Adaptive responses to osmotic stress in kidney-derived cell lines from Scatophagus argus, a euryhaline fish.

The euryhaline fish, the spotted scat (Scatophagus argus), is exceptional for its ability to tolerate rapid fluctuations in salinity. To better understand fish osmoregulation and enable more precise analyses of specific features of adaptive responses to the osmotic stress in fish, a S. argus kidney-derived cell line (SK) was developed and subcultured for more than 70 passages. The cells were mostly fibroblast-like, with a normal diploid karyotype (2n=48). A low-osmolarity-adapted SK cell line (SK-la) was induced by growth in a hypotonic solution (150 mOsm). Effects of different osmotic stresses (150, 300 and 450 mOsm) on cell growth, cell morphology, cell volume changes and cell damage in SK, SK-la and CIK (a kidney-derived cell line from freshwater grass carp) cells were studied. These were compared by use of microscopic observation, flow cytometry and a Na-K-ATPase (NKA) assay. SK cells became smaller and grew rapidly in response to hypotonic stress (150 mOsm), and exhibited no visible morphological changes in response to hypertonic stress (450 mOsm). SK-la grew well by moderate hypertonicity (300 mOsm) but depressed in severe hypertonicity (450 mOsm), the number of unhealthy SK-la cells rose as osmolarity increased. In contrast, CIK cells became unhealthy with anisotonic challenge. The NKA activities of SK and CIK cells were assayed after exposure to anisotonic conditions, and rapid decreases were detected immediately except SK cells which were not affected in hypotonicity. Unlike in SK and CIK, an increase following a down-regulation of NKA activity was observed in SK-la cells upon moderate hypertonic stress. These results suggested that SK and SK-la cells had stronger osmoregulatory capacity than CIK cells, and provided new insights on the osmosensing and osmotic adaption in euryhaline fish kidney.

Histological study on the gonadal development of Scatophagus argus

The fish spotted scat Scatophagus argus,as a new resource of marine aquaculture,is popularized in South China in recent years.Studies on the gonadal development can provide the theoretical knowledge for the artificial propagation of Scatophagus argus.Generally,after two years of growth,some individuals can reach sexual maturation in the south of China.In this study,histological observation of gonads was performed based on paraffin section technique and HE staining method.The linear alignments of tubular structures are observed in the testis,which indicates that the testis of the Scatophagus argus belongs to the tubular type.The development of testis may be divided into 5 stages,i.e.multiplication of spermatogonial cells,growth of spermatocytes,maturation of spermatocytes,emergence of spermatoblasts,maturation of spermatozoa,etc.The average gonado somatic index of the testis was between 0.2% and 1.5%.The gonado somatic index(GSI)reached the peak at the stageⅤ,and the the peak of hepato somatic index(HSI)was observed at the stage Ⅳ.The oocyte deveolpment was divided into 5 phases and ovary development consisted of 5 stages accordingly.The average gonado somatic index of the testis was between 1.2% and 14.5%.The gonado somatic index(GSI)reached the peak value at the stage Ⅴ and the peak of hepato somatic index(HSI)was found in stage Ⅳ.The yolk nucleus and follicle membrane are observed at phase Ⅱ of oocyte.The number of yolk granules and oil droplets increased rapidly at the phaseⅢ,and subsequently yolk granules fused into pieces at the phase Ⅳ Apart from a large number of oocytes at the phase Ⅴ,there are still many oocytes of phase Ⅱ,Ⅲ and Ⅳ in ovaries at the stage Ⅴ.The similar condition was also found in ovaries after spawning.It indicates that oocyte development in Scatophagus argus is asynchronous.

Isolation and characterization of EST-based microsatellite markers for Scatophagus argus based on transcriptome analysis

In the present study, expressed sequence tag-based microsatellite markers were developed by 454 pyrosequencing for the marine fish Scatophagus argus. A total of 510,939 reads were obtained, and 4,322 di-nucleotides, 1,354 tri-nucleotides, 219 tetra-nucleotides, 30 penta-nucleotides and 9 hexa- nucleotides were detected. Among these repeated sequences, 51 microsatellites where repeat units were greater than 4 were randomly selected to test the variability in natural populations. 12 polymorphic markers were identified and characterized. The number of alleles per locus varies from 3 to 6, and observed and expected heterozygosities ranged from 0.102 to 0.658 and from 0.124 to 0.691, respectively. No genetic linkage and significant departure from Hardy–Weinberg equilibrium were detected among these loci. High-throughput pyrosequencing allows fast and efficient isolation of microsatellite markers on a large scale, and can provide enough microsatellite markers for population genetic studies and genetic mapping.

Novel polymorphic microsatellite loci for the spotted scat Scatophagus argus

Scatophagus argus is a recreational and commercial fish in East Asia. There is no genetic information about wild populations available so far. A first set of 15 microsatellite loci were developed from the species and characterized. The number of alleles per locus varied from 3 to 12 with the average of 6.6. Levels of observed heterozygosity (HO) per locus ranged from 0.421 to 0.876, and the expected heterozygosity (HE) per locus was from 0.497 to 0.931. No significant linkage disequilibrium was detected between loci, and no departure from Hardy–Weinberg equilibrium was observed except for the locus SA108. These polymorphic microsatellite markers would aid in the conservation and management in Scatophagus argus.

Dopamine regulates renal osmoregulation during hyposaline stress via DRD1 in the spotted scat (Scatophagus argus).

Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity. However, the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood. We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish Scatophagus argus (S. argus) during abrupt transfer from seawater (SW) to freshwater (FW). Following the transfer, serum dopamine concentration was decreased, and dopamine activated expression of the dopamine receptor 1 (designated SaDRD1) in the kidney, triggering the osmoregulatory signaling cascade. SaDRD1 protein is expressed in the renal proximal tubule cells in vivo, and is localized to the cell membrane of renal primary cells in vitro. Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase (NKA) activity in cultured renal primary cells in vitro, suggesting that expression of SaDRD1 may oppose the activity of NKA. We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium. Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity, providing a new perspective on the hyposaline adaptation of fish.

Complete mitochondrial genome of the striped scat Selenotoca multifasciata (Perciformes: Scatophagidae)

Abstract The striped scat Selenotoca multifasciata is an ornamental and commercial fish in Asia. In the present study, we sequenced and annotated the complete mitochondrial genome of Selenotoca multifasciata. Its total length is 16,646 bp, and the mitochondrial genome is composed of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding control region. ND6 and eight tRNA genes were encoded on the light strand, whereas the remaining genes located on the heavy strand (H-strand). All the 16 mitochondrial genomes of the suborder Acanthuroidei available in GenBank were employed for phylogenetic analysis, and the result showed a close relationship between Selenotoca multifasciata and Scatophagus argus. This mitochondrial information may benefit relative ecological and phylogenetic studies.

Isolation and characterization of 16 polymorphic microsatellite loci in the leopard coral grouper Plectropomus leopardus

Plectropomus leopardus is an important commercial fish in South-East Asia. To date, there is little genetic information available about its population structure. In this study, 16 polymorphic microsatellite loci were developed for this species and characterized in 182 wild individuals. The observed and expected heterozygosity ranged from 0.236 to 0.912 and from 0.649 to 0.866, respectively. No linkage disequilibrium was detected between loci, and four loci were found to significantly depart from Hardy–Weinberg equilibrium. These polymorphic microsatellite markers are valuable for relative studies of population genetics.