Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill) stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.     

Primary molecular basis of androgenic gland endocrine sex regulation revealed by transcriptome analysis in Eriocheir sinensis

In crustaceans, the male sexual differentiation and maintenance are specially regulated by androgenic gland (AG). However, little is known about the genes involved in the regulation process. RNA-Seq was performed on AG with ejaculatory duct (AG_ED) and ejaculatory duct (ED) as control in Eriocheir sinensis, one of the most important economic and fishery crabs with typically sex dimorphism. A total of 925 unigenes were identified as differentially expressed genes (DEGs) and the expression of nine genes randomly selected was confirmed by qRT-PCR. 667 unigenes were up-regulated in AG_ED, being supposed to be AG preferential genes. Among them, the full length of insulin-like androgenic gland factor (IAG) cDNA named as Es-IAG was obtained as a logo gene of AG, which together with the genes insulin-like receptor (INR), and single insulin binding domain protein (SIBD), might constitute the sex regulation pathway. Several sex related genes were identified, and their function will have to be investigated. Also, the identification of juvenile hormone epoxide hydrolase 1 (JHEH1), ecdysteroid 22-hydroxylase (DIB) and ecdysone receptor (ECR) preliminarily clarified the molecular regulation mechanism of eyestalk-AG-testis axis, which plays important roles in molting and reproduction. The results will enhance our understanding for the molecular basis of the AG involved in male sex regulation in crabs.

     

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.     

Effects of temperature and salinity on the development of the amphipod crustacean Eogammarus sinensis

The amphipod crustacean Eogammarus sinensis has useful features that make it suitable for use in the aquaculture of fish and large decapod crustaceans.In this study,we investigated the effects of temperature and salinity on the development,fecundity,survival,and growth rate of E.sinensis.The results show that temperature significantly affected E.sinensis development,but salinity.As temperature increased,the duration of E.sinensis embryonic development decreased.Fecundity was affected significantly by temperature and the combination of temperature and salinity,but by salinity alone.In addition,high temperatures accelerated E.sinensis juvenile growth rates,whereas high salinity reduced it.Therefore,our data suggest that E.sinensis tolerates a wide range of salinities and that temperature has more significant effects than salinity on the embryonic development,fecundity,and growth of E.sinensis.Our results shall be useful for mass production of this species for use in aquaculture.     

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

1 INTRODUCTION The unicellular Dunaliella salina, is one of the well studied microalgae for mass culture and is of commercial importance as a source of natural beta-carotene (Avron and Ben-Amot, 1992). D. sa- lina has desirable properties of halotolerance…     

马氏珠母贝外套膜中央膜与边缘膜荧光定量PCR分析中内参基因的筛选

为筛选能在马氏珠母贝外套膜边缘膜与中央膜区域稳定表达的内参基因,应用荧光定量PCR 技术,对3-磷酸脱氢酶（GAPDH）、β-肌动蛋白（β-actin）、胆色素原脱氨酶（PBGD）、微管蛋白（TUB）、TATA 盒结合蛋白（TBP）、磷脂酶A2（PLA-2）、葡萄糖苷酶（GUSB）、18S 核糖体rRNA（18SrRNA）等8 个常用的内参基因在马氏珠母贝外套膜边缘膜与中央膜区域的表达情况进行分析,并利用geNorm、NormFinder、BestKeeper 及RefFinder 软件对8 个内参基因的表达稳定性进行分析.结果表明：8 个内参基因均可获得特异性扩增产物,但稳定性各异, 其在外套膜边缘膜和中央膜区的表达稳定性顺序依次为PBGD/TBP〉TUB〉GUSB〉18SrRNA〉PLA-2〉GAPDH〉 β-actin;在荧光定量PCR 中,PBGD 和TBP 在马氏珠母贝外套膜边缘膜和中央膜区的表达比较稳定,为研究贝壳矿化机制中理想的内参基因.     

Ecological footprint and major driving forces in West Jilin Province,Northeast China

The environmental impact caused by local people (ecological footprint of consumption, EFc) and the actual environmental impact that the ecosystem burdens (ecological footprint of production, EFp) in West Jilin Province, Northeast China from 1986 to 2006 were evaluated by using ecological footprint (EF) method. And the major driving forces of EFc and EFp were analyzed by STIRPAT model. Both EFc and EFp showed increasing trends in 1986-2006, accompanied by decreasing ecological deficits but expanding ecologic...     

DNA barcoding discriminates Pampus minor (Liu et al., 1998) from Pampus species

Although Pampus minor has been classified as a new species, it still remains controversial. Was used a DNA barcoding technique based on homologous sequence analysis of the16S and COI genes to clarify the confusion over the identification of this species. Among 12 individuals whose genetic distance was 0.002, two haplotypes were found. According to the 16S sequences, the genetic distances ranged from 0.121 to 0.133 between P. minor and other Pampus species. Although the same the genetic distance between the two P. minor haplotypes was generated using COI sequences, the haplotype of Pm22-23, Pm28, and Pm32-33 was the same as that of Pci EF607462 and EF607466, while the haplotype of Pm24-27 and Pm29-31 was the same as that of Pci EF607461 and EF607463-65. In addition, the genetic distance ranged only from 0.002 to 0.005 between P. minor and Pa EF607460 and EF607458. Apart from this, the interspecies genetic distances varied from 0.135 to 0.143 between P. minor and other Pampus species according to the COI sequences. Phylogenetic trees, using combined 16S and COI data, strongly support the viewpoint that all the P. minor individuals form one clade that is in a sister position to Pampus sp. individuals (EU357803, FJ434342-FJ434343, and FJ652423-FJ652427).     

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> in grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.     

Nemerteans of Zhoushan Islands,East China Sea Coast,China I. Enopla: Distromatophynchocoela: Monostilifera: Emplectonemertidae

Two nemerteans of the Zhoushan Islands,Paranemertes sinensis sp. nov. andP. peregrina, are reported in this paper.P. sinensis sp. nov. has the body wall longitudinal musculature anteriorly divided, numerous eyes grouped into four clusters, two pairs of cephalic furrows, rhynchocoel 1/3 to 1/2 of full body length, cephalic glands well developed and posteriorly extending beyond the cerebral ganglia, precerebral septum absent, cerebral sense organs situated in front of brain, three pairs of nephridiopores, and 17–18 proboscis nerves. The project supported by National Natural Science Foundation of China     

Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospira platensis in Escherichia coli

Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.     

Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.     

Expression profiles of sex-related genes in gonads of genetic male Takifugu rubripes after 17β-estradiol immersion

Estradiol treatment during early life stages of tiger puffer Takifugu rubripes induces feminization in genetic males. However, the ovaries in genetic males may revert to testes once estradiol treatment is halted.Therefore studies should investigate molecular mechanisms underlying ovary-to-testis recovery in genetic males after treatment. In the present study, tiger puffer were exposed to 10, and 100 μg/L 17 p-estradiol(E_2)from 15 to 100 days post-hatching(dph), then gonad phenotypes and expression profiles of six sex-related genes(cyp19a,foxl2, dmrtl, amh, sox9a, and sox9b) were characterized after the exposure. Results showed that both 10 and 100 μg/L E_2 induced ovarian development in genetic males at 100 dph. However, all ovaries induced by 10 μg/L E_2 first developed into intersexual gonads and subsequently reverted to testes after the exposure. As for treatment of 100 pg/L E_2, while the rest of the ovaries maintained morphological stability,percentages of intersexual gonads reached 38%-57%, and none were reverted to testes. Increased mRNA levels of cyp19 a,foxl2 and sox9b and decreased mRNA levels of dmrtl, amh, and sox9a were observed during the ovarian development in genetic males. While contrary gene expression profiles were detected during ovary-to-testis transformation. The mRNA levels of all the six genes were increased during the development of intersexual gonads. These results indicated that up-regulation of dmrtl, amh and sox9a is associated with initial ovary-to-intersexual transformation, and suppression of foxl2, cypl9a and sox9b is essential for complete ovary-to-testis recovery in genetic males. This research will help to trace the molecular processes underlying gonadal transformation in teleosts.     

Effects of BPA and E2 on expression profiles of genes related to hypothalamic-pituitary-gonadal axis of half-smooth tongue sole Cynoglossus semilaevis

Endocrine disrupting chemicals (EDCs) are increasingly viewed as persistent pollutants, similar to natural hormones in function. This paper describes the expression profiles of 7 genes (DMRT, VTG, GnRHR, FSHR, CYP17A, CYP19A, and CYP19B) involved in sex steroid synthesis and action as well as sexual development in adult male and female Cynoglossus semilaevis, after exposure to different concentrations of Bisphenol A (BPA) and 17β-estradiol (E2). Both BPA (1, 10, 50, 125, and 250 mg/kg) and E2 (0.5, 5, and 10 mg/kg) induced changes in target gene expression, although the estrogenic effects of E2 as a model estrogen were stronger. Among the 7 genes, VTG, CYP17A and CYP19 responded strongly to BPA or E2 exposure and can thus serve as reference biomarkers for estrogenic EDCs exposure in marine teleosts. These data will provide a window to establish a hypothalamic-pituitary-gonadal model in C. semilaevis to better understand the effect pathways of EDCs.