Chunhua Zhu

Effects of fish oil on ovarian development in spotted scat (Scatophagus argus)

The effects of different concentrations of dietary fish oil (0, 2%, or 6%) on ovarian development in 2-year-old female Scatophagus argus were investigated. The levels of serum sex steroid hormones (estradiol-17β, E2; testosterone, T), protein phosphorus (SPP), and protein calcium (SPC), as well as vitellogenin (vtg) mRNA expression in livers and ovaries were measured. Over the eight week experimental period, oocytes did not develop further and remained at phase III in fish fed with the control diet with no supplement of fish oil. Fish fed with 2% fish oil supplement had oocytes at transition phase from III to IV. Fish fed with 6% fish oil supplement had oocytes at late phase IV. Higher gonadosmatic index, serum E2, SPP, SPC, and liver vtg expression were found in 6% fish oil group compared to that in the 2% fish oil group (except E2) and the control group (P<0.05). In addition, vtg expression in livers was 600-1000 times higher than that in the ovaries. Gonadosmatic index, E2, and SPP, as well as liver vtg expression increased during the experiment and peaked at the end of experiment. However, hepatosomatic index, serum T, and ovarian vtg expression peaked at 4 weeks, and then decreased at 8 weeks, with no significant difference among the 3 groups. In summary, we showed that 6% fish oil supplementation in S. argus could effectively promote ovarian development, with associated increases in E2 secretion and increased liver vtg mRNA expression.

Effects of temperature and fish oil supplementation on ovarian development and foxl2 mRNA expression in spotted scat Scatophagus argus

In this study, the complete foxl2 complementary (c)DNA sequence was isolated by simple modular-architecture research tool (SMART)er rapid amplification of cDNA ends (RACE). Two year-old female spotted scat, Scatophagus argus, were reared at different temperatures (23, 26 and 29° C) for 6 weeks, or fed with different concentrations of dietary fish oil (0, 2 or 6%) for 8 weeks. Ovarian development, serum oestradiol-17β (E2 ) levels, as well as ovarian foxl2 expression were measured. At the end of experiment, ovarian foxl2 messenger (m)RNA expression in fish reared at 23 and 26° C was significantly higher than that in fish reared at 29° C, and that in 2 and 6% fish oil groups was also significantly higher than that in control group (P < 0·05). Serum E2 levels exhibited the same trend with foxl2 mRNA expression in temperature treatment groups and fish oil fed groups. There was a significant positive correlation between stage of oocytes and foxl2 expressions. Results showed that from 23 to 29° C, the optimal temperature for ovarian development in S. argus was 23-26° C, and 6% fish oil supplementation could effectively promote ovarian development. Optimal temperature and fish oil supplement might increase ovarian foxl2 mRNA expressions to promote ovarian development in S. argus.

Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus

Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.

Structural and functional characterization of neuromedin S in the teleost fish, zebrafish (Danio rerio)

Neuromedin S (NMS) has been demonstrated to have important roles in many vertebrate physiological processes. However, the function of NMS in teleost fishes remains unclear. We explored the physiological roles of the NMS gene in the zebrafish model. An NMS cDNA was cloned from zebrafish brain tissue, and the full-length cDNA sequence was 521 bp in length and encoded a precursor of 110 amino acid residues. Interestingly, fish prepro-NMS is predicted to generate a short 34-residue peptide, designated as NMS-related peptide (NMSRP). Zebrafish prepro-NMS does not contain the NMS peptide which is found in the NMS precursors of mammals, and just retains the MNSRP peptide. A multiple-species sequence alignment showed that NMSRPs are conserved among the other sampled vertebrates. Zebrafish NMS mRNA was detected by RT-PCR revealing a tissue-specific distribution with high levels of expression in the brain, spleen, ovary, pituitary, and muscle. Furthermore, the locations of NMS-expressing cells in the zebrafish brain were detected by in situ hybridization in the parvocellular preoptic nucleus (PPa), the ventral zone of the periventricular hypothalamus (Hv), and lateral hypothalamic nucleus (LH). The levels of NMS mRNA in the hypothalamus were significantly increased after three days of food deprivation. Administration of zebrafish NMSRP by intraperitoneal injection significantly promoted the expression of neuropeptide Y (NPY) and orexin, suggesting an orexigenic role for NMSRP in zebrafish. The present study offers a new understanding of the NMS gene in vertebrates and increases our knowledge of the neuroendocrine regulation of feeding.

Cloning, expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus

Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erβ1 and erβ2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erβ1 and erβ2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erβ1 and erβ2 were not, with three vtgs in female liver. The effects of 17β-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erβ1, vtgs mRNA and Vtg protein increased significantly after 24h incubation with E2 (0.1, 1 and 10μM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1μM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1μM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erβ1 and vtg-A. Erβ selective antagonist Cyclofenil (0.01, 0.1 and 1μM) attenuated the up-regulation effects of E2 on erβ1, erβ2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.

Characterization of the complete mitochondrial genome of Priacanthus tayenus (Perciformes: Priacanthidae) with phylogenetic consideration

Abstract The complete mitochondrial genome of the Priacanthus tayenus was sequenced and analyzed in this study. The mitochondrial genome is 16,866 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and the composition of P. tayenus mitochondrial genome were similar to that of most other vertebrates. The nucleotide composition of the light strand in descending order is 30.91% of G, 26. 35% of T, 25.61% of A and 17.13% of C. The NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes were localized in the light strand, and all other mitochondrial genes were encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method revealed that the P. tayenus showed the closer relationship to the Sciaenops ocellatus.

Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus (Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs

Spotted scat (Scatophagus argus) is an economically important farmed fish, particularly in East and Southeast Asia. Because there has been little research on reproductive development and regulation in this species, the lack of a mature artificial reproduction technology remains a barrier for the sustainable development of the aquaculture industry. More genetic and genomic background knowledge is urgently needed for an in-depth understanding of the molecular mechanism of reproductive process and identification of functional genes related to sexual differentiation, gonad maturation and gametogenesis. For these reasons, we performed transcriptomic analysis on spotted scat using a multiple tissue sample mixing strategy. The Illumina RNA sequencing generated 118 510 486 raw reads. After trimming, de novo assembly was performed and yielded 99 888 unigenes with an average length of 905.75 bp. A total of 45 015 unigenes were successfully annotated to the Nr, Swiss-Prot, KOG and KEGG databases. Additionally, 23 783 and 27 183 annotated unigenes were assigned to 56 Gene Ontology (GO) functional groups and 228 KEGG pathways, respectively. Subsequently, 2 474 transcripts associated with reproduction were selected using GO term and KEGG pathway assignments, and a number of reproduction-related genes involved in sex differentiation, gonad development and gametogenesis were identified. Furthermore, 22 279 simple sequence repeat (SSR) loci were discovered and characterized. The comprehensive transcript dataset described here greatly increases the genetic information available for spotted scat and contributes valuable sequence resources for functional gene mining and analysis. Candidate transcripts involved in reproduction would make good starting points for future studies on reproductive mechanisms, and the putative sex differentiation-related genes will be helpful for sex-determining gene identification and sex-specific marker isolation. Lastly, the SSRs can serve as marker resources for future research into genetics, marker-assisted selection (MAS) and conservation biology.

Leptin receptor overlapping transcript: characterization, tissue distribution and changes in gene expression in response to eyestalk ablation in the Pacific white shrimp Litopenaeus vannamei

The leptin receptor overlapping transcript (LEPROT), which arose evolutionarily before the leptin receptor, is involved in the nutritional regulation of metabolism and reproduction. Eyestalk ablation is performed on brood stock to induce rapid gonad development in the Pacific white shrimp Litopenaeus vannamei. To clarify the role of L. vannamei leprot (Lvleprot) in gonadal development, Lvleprot cDNA was isolated and a phylogenetic tree was constructed based on the LEPROT amino acid sequences of shrimp, other invertebrates and vertebrates. The resulting topology clearly reflected the taxonomic relationships of the selected organisms. Alignment of LEPROT amino acid sequences showed that there was very high similarity between LvLEPROT and LEPROT from other invertebrates. Quantitative polymerase chain reaction showed that Lvleprot expression was highest in the gonads and intestine, moderate in the hepatopancreas, androgenic gland and muscle, and lowest in gill, heart and stomach, without a sexually dimorphic pattern difference between males and females. After eyestalk ablation, the gonadosomatic index increased significantly, but the hepatosomatic index decreased significantly. In addition, Lvleprot transcript levels in the gonads and intestine decreased significantly, but were significantly increased in the hepatopancreas after eyestalk ablation. These results indicate the significant roles of Lvleprot in the nutritional regulation of reproduction during the period of rapid development in Pacific white shrimp.

The complete mitochondrial genome of the Piaractus brachypomus (Characiformes: Characidae)

Abstract The complete mitochondrial genome of the Piaractus brachypomus is described in the present study. The mitochondrial genome is 16,561 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The P. brachypomus mitochondrial genome shows the similar gene order and composition with those of most other vertebrates. The nucleotide compositions of the light strand in descending order is 31.57% of A, 26.19% of C, 26.18% of T and 16.06% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand.

Illumina next-generation sequencing reveals the complete mitochondrial genome of Psenopsis anomala (Perciformes: Centrolophidae) with phylogenetic consideration

Abstract Using Illumina next-generation sequencing (NGS), the complete mitochondrial genome of the Psenopsis anomala was sequenced in the present study. The mitochondrial genome of P. anomala is 16,528 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The structure about gene order and composition of P. anomala mitochondrial genome is similar to those of most other vertebrates. The nucleotide compositions of the light strand in descending order is 29.18% of T, 27.97% of G, 27.06% of A, and 15.79% of C. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method shown that the Psenopsis anomala was closer to Peprilus triacanthus in the phylogenetic relationship.