Jifang Li

The physiological performance and immune responses of juvenile Amur sturgeon (Acipenser schrenckii) to stocking density and hypoxia stress.

Stocking density and hypoxia are considered priority issues in aquaculture research. In this study, two experiments were carried out in order to investigate the effects of chronic stress (stocking density) and acute stress (hypoxia) on the immune physiology responses (hematology, serum cortisol, glucose, total protein and the mRNA expression of CYP 1A) of juvenile Amur sturgeon (Acipenser schrenckii). In the chronic stress study, three triplicate groups of Amur sturgeon (42.0 ± 2.3 g) were reared in nine square concrete ponds (4.4 × 4.4 × 0.45 m³) at three stocking densities (3.7, 6.9 and 9.0 kg/m³) for 50 days. In the acute stress study, three triplicate groups: normal group (7 mg/l), hypoxia group 1 (5 mg/l) and hypoxia group 2 (3 mg/l) were used in nine 100 L indoor tanks. Sampling was performed at the end of the stocking density experiment (50 days) and at 0, 0.5, 1.5, 3 and 6 h after hypoxia stress. The results showed that increased stocking density reduced the morphological indexes (hepatosomatic index, spleen-somatic index and kidney-somatic index), while total protein and hemoglobin increased significantly in the stressed group. In response to hypoxia, the levels of cortisol, glucose and hematological parameters elevated significantly after this stress. As for spleen-somatic index, there was a decline after hypoxia though H1 group returned to the normal level at 3 h and 6 h after hypoxia stress. Additionally, In order to better understand the immune response of Amur sturgeon to chronic and acute stressors, we cloned the complete coding sequence of Amur sturgeon CYP 1A for the first time and investigated its tissue-specific expression and stress-induced expression. CYP 1A mRNA in liver showed over expressions both in crowding condition and in hypoxia stress. The same trend was also found in spleen and kidney which may provide evidence that CYP 1A could serve as a good indicator of immune response in Amur sturgeon. In addition, the result suggested a typical immune response both in high stocking density and hypoxia stress. But the chronically stressed fish might have an adaptation capability to survive under a stable crowding condition without a change in some immune parameters (cortisol, glucose, WBCs and RBCs).

DNA methylation level of cyp19a1a and Foxl2 gene related to their expression patterns and reproduction traits during ovary development stages of Japanese flounder (Paralichthys olivaceus)

Foxl2 and cyp19a1a genes are crucial for the ovarian development, and Foxl2 could play a direct regulatory role on cyp19a1a transcription. In this study, we aimed to study DNA methylation status and mRNA expression patterns of Foxl2 and cyp19a1a genes during ovarian development of female Japanese flounder. The relative expression level of cyp19a1a and Foxl2 gene during the gonadal development stages was measured by quantitative PCR. Moreover, DNA methylation status in the promoter and coding regions of the two genes was detected by bisulfite sequencing. The estradiol-17β (E2) was measured by radioimmunoassay. The results showed low expression levels of cyp19a1a and Foxl2 genes in stages II and V, while the highest expression levels were detected in stage IV. The variation trend of the methylation level of all CpG sites in promoter and exon 1 of cyp19a1a gene and three CpG rich regions in coding region of Foxl2 gene was negatively associated with their expression levels during the ovarian development. In addition, two CpG sites in promoter and seven CpG sites in exon 1 of cyp19a1a were on the putative transcription factors binding sequence. Further studies showed that the forkhead domain, which is important for Foxl2 binding to cyp19a1a was located in the F1 and F2 region. These results provide a powerful theoretical basis for the regulatory mechanism on Foxl2 regulating cyp19a1a and promoting gonadal differentiation towards the female pathway, and further reveal that Foxl2 and cyp19a1a play a vital role in the female Japanese flounder gonad development.

DNA methylation status of cyp17-II gene correlated with its expression pattern and reproductive endocrinology during ovarian development stages of Japanese flounder (Paralichthys olivaceus).

Cytochrome P450c17-II (cyp17-II, 17α-hydroxylase) is responsible for the production of steroid hormones during oocyte maturation in vertebrates. The comparative expression pattern of cyp17-II gene during the gonadal development stages will provide important insights into its function of gonadal development. In addition, epigenetic modification especially DNA methylation plays a vital role in regulation of gene expression. The adult female Japanese flounder at different ovarian development stage (from stages II to V) was obtained in this experiment. The expression of cyp17-II gene in the ovary of Japanese flounder during the gonadal development stages was measured by quantitative PCR. Reproductive traits included gonadosomatic index (GSI), plasma estradiol-17β (E2) and testosterone (T) were also measured. Moreover, whole CpG dinucleotides methylation status of the two CpG rich regions in cyp17-II coding region was detected by bisulfate sequencing. In the ovary, the cyp17-II gene had the lowest mRNA expression at the early ovarian development stage, but then increased afterward. The variation trends of T and E2 level were consistent with the cyp17-II expression pattern in ovary. In contrast, the whole methylation levels of each CpG rich region (exon 4 and 6) in cyp17-II coding region were declined from stages II to IV, then increased at stage V. The methylation levels of whole CpG sites in each CpG rich region were inversely correlated with the values of ovarian cyp17-II gene expression, T and E2 level, and GSI. Based on the present study, we proposed that cyp17-II may regulate the level of steroid hormone, and then stimulate the oocyte growth and maturation. The cyp17-II gene transcriptional activity was possibly affected by the methylation level of CpG rich regions in coding region. These findings will help in the study of the molecular mechanism of fish reproduction and endocrine physiology.

Polymorphism in exons CpG rich regions of the cyp17-II gene affecting its mRNA expression and reproductive endocrine levels in female Japanese flounder (Paralichthys olivaceus)

Cytochrome P450c17-II (cyp17-II) gene is an important factor affecting the growth, gonad differentiation and development, and other reproductive traits of fish. There are three CpG rich regions in the coding region of cyp17-II gene in Japanese flounder (Paralichthys olivaceus). The aim of this study was to understand whether mutations in exons of the cyp17-II gene occured at CpG sites, and mutations and methylation status of those CpG sites were involved in regulation of the expression level of cyp17-II gene and the reproductive endocrine of Japanese flounder. The results showed that three single nucleotide polymorphisms (SNPs) were identified. SNP1 [(c. G594A (p.Gly 188Arg)] located in exon 4 of L1 locus, and SNP2 (c.A939G) and SNP3 (c.C975T) of L2 locus located in CpG rich region of the exon 6 of cyp17-II gene. Furthermore, the A to G transition at 939bp position added a new methylation site to the cyp17-II coding region. According to multiple-comparison analysis, two loci (L1 and L2) were significantly associated with serum testosterone (T) level (P<0.05) and the expression of cyp17-II in ovary (P<0.01). Intriguingly, individuals with GG genotype of L2 locus containing eight CpG methylation sites had significantly lower serum testosterone level and cyp17-II mRNA expression than those with AA genotype containing seven CpG methylation sites. Moreover, the CpG site was highly methylated (≥77.8%) at 938 bp position of individuals with GG genotype of L2 locus. These implied that the mutation and methylation status of the coding region of cyp17-II could influence the gene expression and the reproductive endocrine levels in female Japanese flounder and L2 locus could be regarded as a candidate genetic or epigenetic marker for Japanese flounder breeding programs.

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli).

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.

Effect of stocking density on water quality and (Growth, Body Composition and Plasma Cortisol Content) performance of pen-reared rainbow trout ( Oncorhynchus mykiss )

The goal of the study was to examine the effect of stocking density on the water quality of culture area, as well as the growth, body composition and cortisol content of rainbow trout (Oncorhynchus mykiss). Pen-reared trout were stocked in densities of 40, 60, 80 fish individuals m−3 (4.6, 6.6, 8.6 kg m−3, SD1, SD2 and SD3 groups, respectively) for 300 days. Compared to the water from SD1 and SD2, that from SD3 exhibited significantly higher NH4+-N content and COD (chemical-oxygen-demand), and a significant reduction of dissolved oxygen in day 180 (40.6 kg m−3). Stocking density was significantly associated with body weight, standard length, VSI (viscerosomatic index), CF (condition factor) and FC (food coefficient) in group SD3, particularly in day 240 and day 300 (45 or 49.3 kg m−3). Increased crude fat and decreased crude protein were displayed in high density group when the density reached to 36 kg m−3. As a cumulative effect of density-related stress, VSI, CF, FC, moisture, and crude protein content varied over time in each density group (SD1, SD2, and SD3). In summary, trout exhibited a better growth performance in low density (26.3 kg m−3) than those reared in high densities (36 and 45 kg m−3). The results indicate that rainbow trout (114.44 g ± 6.21 g, 19.69 cm ± 0.31 cm) initially stocked in 6.6 or 8.6 kg m−3 should be lightened to less than 36 kg m−3 after an intensive rearing for 240 days.

Expression Analysis of the Insulin-Like Growth Factors I and II During Embryonic and Early Larval Development of Turbot （Scophthalmus maximus）

The insulin-like growth factors I and II (IGF-I and IGF-II) are important proteins involved in fish growth and development. Here, we report the isolation of IGF-II and expression analysis of IGFs in turbot Scophthalmus maximus, aiming to clarify their function in embryonic and larval development of fish. The deduced IGF-II gene is 808 bp in full length, which encodes a protein of 219 amino acids and is 93% similar with that of Paralichthys olicaceus in amino acid sequence. The tissue abundance and the expression pattern of IGFs in a turbot at early development stages were investigated via reverse transcription-polymer chain reaction. Result showed that the IGF-I and IGF-II genes were widely expressed in tissues of S. maximus. IGF-I was detected in all tissues except intestines with the highest level in liver, while IGF-II transcript presented in all tissues except muscle. At the stages of embryonic and larval development, the mRNA levels of IGFs sharply increased from the stage of unfertilized egg to post larva, followed by a decrease with larval development. However, there was an increase in IGF-I at the embryonic stage and IGF-II at the gastrula stage, respectively. These results suggested that IGFs play important roles in cell growth and division of the turbot. Our study provides reference data for further investigation of growth regulation in turbot, which can guarantee better understanding of the physiological role that IGFs play in fish.

Cloning and expression analysis of vasa during the reproductive cycle of Korean rockfish, Sebastes schlegeli

Vasa, which is a conserved member of the DEAD-box protein family, plays an indispensable role in primordial germ cell proliferation. However, the expression of vasa gene during the reproductive cycle in ovoviviparous fish has not been documented. In this study, the full-length sequence of vasa was obtained from the ovary of Korean rockfish (Sebastes schlegeli) using reverse transcription-PCR and rapid amplification of cDNA ends. The Vasa with a mature protein of 650 amino acids showed greatest homology (84%) with giant gourami (Osphronemus goramy) and Pacific bluefin tuna (Thunnus orientalis). The expression of vasa mRNA in Korean rockfish was detected in gonads only, suggesting its specific role in gonadal development. In addition, seasonal changes in the vasa expression levels were examined in gonads by quantitative real-time PCR. The vasa transcript levels in adult testis were found higher during spermatogenesis than during spermiation. The vasa transcript levels remained relatively high at the early ovary stage but declined during ovary maturation in adult female fish. These results suggest that the vasa gene play an important role in spermatogenesis and early oogenesis during the reproductive cycle of Korean rockfish.

Analysis of DNA methylation level by methylation-sensitive amplification polymorphism in half smooth tongue sole (Cynoglossus semilaevis) subjected to salinity stress

Increasingly arisen environmental constraints may contribute to heritable phenotypic variation including methylation changes, which can help the animals with development, growth and survival. In this study, we assessed the DNA methylation levels in three tissues (gonad, kidney and gill) of half smooth tongue sole under the salinity stress. The methylation-sensitive amplification polymorphism (MSAP) technique was applied to illustrate the regulation of epigenetic mechanism in environmental stimuli. Fish were subjected to 15 salinity treatment for 7 and 60 days, respectively. A total of 11259 fragments were amplified with 8 pairs of selective primers. The levels of methylated DNA in different tissues of females and males without salinity stress were analyzed, which were 32.76% and 47.32% in gonad; 38.13% and 37.69% in kidney; 37.58% and 34.96% in gill, respectively. In addition, the significant difference was observed in gonad between females and males, indicating that discrepant regulation in gonadal development and differentiation may involve sex-related genes. Further analysis showed that total and hemi-methylation were significantly decreased under 15 salinity for 7 days, probably resulting in up-regulating salt-tolerance genes expression to adjust salt changing. With the adjustment for 60 days, total and hemi-methylation prominently went back to its normal levels to obtain equilibrium. Particularly, full methylation levels were steady along with salinity stress to maintain the stability of gene expression. Additionally, the data showed that gonads in females and gills in males were superior in adaptability. As a result, DNA methylation regulates tissue- specific epiloci, and may respond to salinity stress by regulating gene expression to maintain animal survival and activity.

Expression of estrogen receptors in female rainbow trout (Oncorhynchus mykiss) during first ovarian development and under dense rearing condition

To study the expression of four estrogen receptor genes (erα1, erα2, erβ1, erβ2) of female rainbow trout (Oncorhynchus mykiss) during first ovarian development, trouts were sampled from different ovarian stages. Serum E2 (estradiol) was measured by ELISA and estrogen receptors mRNA expression were examined by qRT-PCR. Our results showed a close association between increased erα1 and vitellogenin mRNA expression during ovarian maturation and increased erα2 mRNA expression in mature ovarian stages. Correlation analysis revealed that a negative relationship between serum E2 and ovarian erβ1 (or hepatic erβ2), but ovarian erβ2 mRNA expression was relatively unchanged during first ovarian development. Trout were also reared in different densities as stocking density 1, 2 and 3 (SD1, 4.6-31.1 kg/m3; SD2, 6.6-40.6 kg/m3; SD3, 8.6-49.3 kg/m3) to elucidate effects of high density on estrogen receptor expression. Histology observation showed ovarian development of trout in higher densities were retard with a relatively early stage and fewer vitellogenin accumulation. Trout in high densities showed significantly decreased serum E2, erα mRNA expression and increasing trends of erβ mRNA expression. A noticeable increase of ovarian erβ2 mRNA expression was seen in trout when density is approaching to 50 kg/m3. In conclusion, we may hypothesize that increased erβ mRNA expression triggered by high density result in decreased erα mRNA expression and vitellogenesis. As a result, ovarian development in higher densities was retard.