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.     

Analysis of DNA Methylation Level of Portunus trituberculatus Subjected to Low Salinity with Methylation-Sensitive Amplification Polymorphism

In this study, the methylation-sensitive amplification polymorphism(MSAP) was used to compare the genomic DNA methylation level of muscle, gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation. Thirty primers were used to analyze the difference of methylation level of different tissues. The results showed that the baseline methylation level of muscle, hepatopancreas and gill was 47.31%, 22.94% and 17.69%, respectively. After exposed to low salinity stress, the methylation epiloci changed in the three tissues. Both demethylation and methylation processes occurred under low salinity stress. The methylation ratio decreased in muscle and gill but increased in hepatopancreas. These results indicated that DNA methylation is tissue-specific when P. trituberculatus responds to low salinity.     

DNA Methylation of pacap Gene Is Involved in Growth Superiority of Female Half Smooth Tongue Sole(Cynoglossus semilaevis) in Different Salinities

Pituitary adenylate cyclase activating polypeptide(PACAP)and growth hormone-releasing hormone(GHRH)play important roles in the GH/IGF growth axis in fishes.To determine whether epigenetic change is involved in the regulation of pacap and ghrh responses to low salinity stress in Cynoglossus semilaevis,the correlation between growth traits,DNA methylation status and gene expression level in low salinity(15,S15)and optimal salinity(30,S30)at day 7(D7)and day 60(D60)were analyzed.Results showed that exposure to low salinity for 60 days attenuated C.semilaevis growth rate.Under low salinity,DNA methylation level of pacap promoter increased in females and decreased in males at day 7,but inverted at day 60.Additionally,pacap expression was up-regulated in both males and females.The pacap promoter methylation level was higher and expression level was lower in female than in male.The results suggest that pacap promoter methylation level is negatively correlated to mRNA level and positively correlated to body weight,while gene expression level is negatively related with body weight.With the decrease of salinity,DNA methylation level of ghrh promoter and exon1,as well as its gene expression displayed minor changes.Overall,pacap gene seems to play an important role in fish growth,contributing to female growth superiority,while ghrh gene seems not pertinent under salinity stress.The results indicate that low salinity potentially affects fish growth through regulating DNA methylation in pacap promoter.This study expands the understanding of the molecular mechanism of how salinity modulates fish growth from the epigenetic perspective.     

Half Smooth Tongue Sole(Cynoglossus semilaevis) Under Low Salinity Stress Can Change Hepatic igf2 Expression Through DNA Methylation

Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand the epigenetic regulation mechanism of igf2 under low salinity stress,we analyzed the DNA methylation at 5’UTR,exon1,intron1,and exon2,and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity(15)for 0,7,and 60 d.When exposed to low salinity,DNA methylation at 5’UTR and exon2 remained stable,while it was up-regulated firstly and then down-regulated at exon1 and intron1.Some single CpG sites of igf2 associated with low salinity,and most of these sites with significantly changed methylation levels(P<0.05)are located in intron1 area.The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity.Remarkably,the 38-CpG site of intron1 servers as a sexual marker.Additionally,our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression.The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress.Above all,to resist the low salinity stress,half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.     

Effects of salinity on egg and fecal pellet production,development and survival,adult sex ratio and total life span in the calanoid copepod, <Emphasis Type="Italic">Acartia tonsa</Emphasis>: a laboratory study

The effects of salinity on the copepod, Acartia tonsa in terms of daily egg production rate (EPR), hatching success, fecal pellet production rate (FPR), naupliar development time and survival, sex ratio, and total life span were determined in laboratory conditions through three experiments. In experiment 1, EPR, hatching success, and FPR of individual females were monitored at salinities of 13, 20, 35 and 45 during short-periods (seven consecutive days). Results show EPR was affected by salinity with the highest outputs recorded at 20 and 35, respectively, which were considerably higher than those at 13 and 45. Mean FPR was also higher in 35 and 20. In experiment 2, the same parameters were evaluated over total life span of females (long-term study). The best EPR and FPR were observed in 35, which was statistically higher than at 13 and 20. In experiment 3, survival rates of early nauplii until adult stage were lowest at a salinity of 13. The development time increased with increasing of salinity. Female percentage clearly decreased with increasing salinity. Higher female percentages (56.7% and 52.2%, respectively) were significantly observed at two salinities of 13 and 20 compared to that at 35 (25%). Total longevity of females was not affected by salinity increment. Based on our results, for mass culture we recommend that a salinity of 35 be adopted due to higher reproductive performances, better feeding, and faster development of A. tonsa.     

鞍带石斑鱼人工繁殖及胚胎发育研究

为建立鞍带石斑鱼(Epinephelus lanceolatus)人工繁殖技术,研究了鞍带石斑鱼亲鱼培育、催产及胚胎发育过程。根据性腺检查及催产结果,判定鞍带石斑鱼在广东的繁殖季节为每年的7-9月份,产卵水温在28~31℃。使用10~12尾雌鱼和3尾雄鱼搭配进行催产实验,亲鱼未出现发情和追尾现象,虽能产卵但均未受精,通过人工授精可得受精卵。对鞍带石斑鱼胚胎发育进行了观察,详细描述从受精卵到初孵仔鱼的28个具体发育时期的形态特征和发育时间,结果表明,将鞍带石斑鱼胚胎发育划分为卵裂期、囊胚期、原肠胚期、神经胚期和器官形成期。在水温(27±0.5)℃、盐度31.0、pH 7.8的海水中,鞍带石斑鱼胚胎历时25 h 40 min完成整个胚胎发育孵化出膜。     

Effect of Salinity on Hemolymph Osmotic Pressure, Sodium Concentration and Na^＋-K^＋-ATPase Activity of Gill of Chinese Crab, Eriocheir sinensis

The effects of salinity on hemolymph osmotic pressure, Na^＋ concentration and Na^＋-K^＋-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na^＋ concentration increased significantly （P〈0.05）, and the Na^＋-K^＋-ATPase activity of gills decreased significantly （P〈0.05） when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na^＋ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na^＋-K^＋-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na^＋ concentration and lower Na^＋-K^＋-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct （P〉0.05）; However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1d, and then tended to be stable from day 1 to day 15.     

Effects of temperature and salinity on survival,growth and DNA methylation of juvenile Pacific abalone,Haliotis discus hannai Ino

Temperature and salinity are two of the most potent abiotic factors infl uencing marine mollusks.In this study,we investigated the individual and combined ef fects of temperature and salinity on the survival and growth of juvenile Pacifi c abalone,Haliotis discus hannai Ino,and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to dif ferent rearing conditions.The single-factor data showed that the suitable ranges of temperature and salinity were 16–28°C at a constant salinity of 32,and 24–40 at a constant temperature of 20°C,respectively.The two-factor data indicated that both survival and growth were signifi cantly af fected by temperature,salinity and their interaction.The optimal temperature-salinity combination for juveniles was 23–25°C and 30–36.To explore environment-induced DNA methylation alteration,the methylation-sensitive amplifi ed polymorphism(MSAP)technique was used to analyze the genomic methylation profi les of abalone reared in optimal and adverse conditions.Neither temperature nor salinity induced evident changes in the global methylation level,but 67 and 63 dif ferentially methylated loci were identifi ed in temperature and salinity treatments,respectively.The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic dif ferentiation in H.discus hannai Ino.The results of our study provide optimal rearing conditions for juvenile H.discus hannai Ino,and represent the fi rst step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.     

Seasonal variation of biochemical components in clam (<Emphasis Type="Italic">Saxidomus purpuratus</Emphasis> Sowerby 1852) in relation to its reproductive cycle and the environmental condition of Sanggou Bay,China

Seasonal variation of biochemical components in clam (Saxidomus purpuratus Sowerby 1852) was investigated from March 2012 to February 2013 in relation to environmental condition of Sanggou Bay and the reproductive cycle of clam. According to the histological analysis, the reproductive cycle of S. purpuratus includes two distinctive phases: a total spent and inactive stage from November to January, and a gametogenesis stage, including ripeness and spawning, during the rest of the year. Gametes were generated at a low temperature (2.1°C) in February. Spawning took place once a year from June to October. The massive spawning occurred in August when the highest water temperature and chlorophyll a level could be observed. The key biochemical components (glycogen, protein and lipid) in five tissues (gonad, foot, mantle, siphon and adductor muscle) were analyzed. The glycogen content was high before gametogenesis, and decreased significantly during the gonad development in the gonad, mantle and foot of both females and males, suggesting that glycogen was an important energy source for gonad development. The protein and lipid contents increased in the ovary during the gonad development, demonstrating that they are the major organic components of oocytes. The lipid and protein contents decreased in the testis, implying that they can provide energy and material for spermatogenesis. The results also showed that protein stored in the mantle and foot could support the reproduction after the glycogen was depleted.     

Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>) tissues

Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass (Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

Salinity tolerance and growth response of juvenile <Emphasis Type="Italic">Oreochromis mossambicus</Emphasis> at different salinity levels

1　Introduction　Theuseofnaturalresources ,includingthosebeingofseeminglymarginalvalue ,isanimportanthumanactivitydesignedtoincreasefoodproductionandin come .Inagriculture ,soilsmaybetoosalinetosup portprofitablecrophusbandry ,yetsuchsoilmaybeusedalternat…     

Effects of salinity on egg and fecal pellet production,development and survival,adult sex ratio and total life span in the calanoid copepod,A cartia tonsa:a laboratory study

The ef fects of salinity on the copepod, A cartia tonsa in terms of daily egg production rate(EPR), hatching success, fecal pellet production rate(FPR), naupliar development time and survival, sex ratio, and total life span were determined in laboratory conditions through three experiments. In experiment 1, EPR, hatching success, and FPR of individual females were monitored at salinities of 13, 20, 35 and 45 during short-periods(seven consecutive days). Results show EPR was aff ected by salinity with the highest outputs recorded at 20 and 35, respectively, which were considerably higher than those at 13 and 45. Mean FPR was also higher in 35 and 20. In experiment 2, the same parameters were evaluated over total life span of females(long-term study). The best EPR and FPR were observed in 35, which was statistically higher than at 13 and 20. In experiment 3, survival rates of early nauplii until adult stage were lowest at a salinity of 13. The development time increased with increasing of salinity. Female percentage clearly decreased with increasing salinity. Higher female percentages(56.7% and 52.2%, respectively) were signifi cantly observed at two salinities of 13 and 20 compared to that at 35(25%). Total longevity of females was not af fected by salinity increment. Based on our results, for mass culture we recommend that a salinity of 35 be adopted due to higher reproductive performances, better feeding, and faster development of A. tonsa.     

Effects of Salinity Fluctuation Frequency on the Osmolarity, Na^＋-K^＋ -ATPase Activity and HSP70 Expression in Juvenile Chinese Shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8...     

盐度突变对青蛤(Cyclinasinensis)鳃Na~+/K~+-ATPase活性的影响

研究了盐度23下青蛤(Cyclina sinensis)鳃、斧足、外套膜和内脏团中Na+/K+-ATPase的活性差异,并以活性高的鳃组织为对象,对青蛤实施了低盐(23→16)和高盐(23→36)突变的胁迫,监测了鳃组织中Na+/K+-ATPase活性的动态变化。结果表明,盐度23下青蛤鳃组织中Na+/K+-ATPase的活性显著高于其他组织;在盐度突降胁迫下,鳃组织中Na+/K+-ATPase的活性于前16 d逐渐升高,第17d起显著升高(p<0.05),第18 d达到峰值,于第20 d回落,但仍高于起始水平;在盐度骤升胁迫下,鳃组织中Na+/K+-ATPase的活性于前13d逐渐降低,第14 d起显著降低(p<0.05),于第15 d达到最低值,继而回升至平稳水平,但仍低于起始水平。青蛤不同盐度胁迫下其鳃组织中Na+/K+-ATPase活性的响应方式不同,低盐时活性增加,而高盐时活性下降;此外,青蛤在盐度胁迫下对自身渗透压的调节则需较长时间。     

Estimation of age,growth and maturation of purpleback flying squid, <Emphasis Type="Italic">Sthenoteuthis oualaniensis</Emphasis>, in Bashi Channel,central Pacific Ocean

The age, growth and maturation of Sthenoteuthis oualaniensis were determined with statolith data collected with a light purse seine from the Bashi Channel of central Pacific Ocean. The estimated longevity of the squid was no more than 6 months for females, and no more than 5 months for males. Growth in mantle length (ML) was best described by logistic models for both females and males, while growth in body weight (BW) was best fitted by power curves. The maximum absolute growth rate (AGR) and instantaneous growth rate (IGR) in ML or BW both occurred at 91–105 days for females and 76–90 days for males. Back calculated hatching dates were from October to January, with a peak in December, although the short duration of sampling date might have had an influence on the result. The lower percentage of mature females (37.2%) suggested that the study area during the sampling date was not a spawning ground for the species. Size and age at first maturity were 183 mm ML and 136 days for females, whereas they were 156 mm ML and 85 days for males.     

Screening of stable internal reference genes by quantitative real-time PCR in humpback grouper Cromileptes altivelis

Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV) through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.