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The light spectrum is a key environmental cue involved in growth and reproduction in teleosts. This study investigated the effects of exposure on juvenile red spotted grouper exposed to white (control), red (590 nm), blue (480 nm), and green (520 nm) light-emitting diodes (LEDs) (12 h light:12 h dark) for two months. The body weight (BW), total length (TL), condition factor (CF), weight gain rate (WGR), gonadosomatic index (GSI), and hepatosomatic index (HSI) were assessed. Gonadal development was observed. The gene expression of growth-related hormones, such as growth hormone (GH), pre-pro-somatostatin-I (PSS-I), neuropeptide Y (NPY), and CCK, and of reproduction-related hormones, such as Kiss1, Kiss2, GPR54, sbGnRH, FSHβ, and LHβ, was analyzed. The results showed that the fish in the white LED group exhibited the best BW, TL, CF, WGR, and HSI after one or two months. The fish exposed to white LEDs showed the best growth after two months, but no significant differences in GH levels were detected. Contrarily, the expression levels of the PSS-I significantly increased (p < 0.05) in fish from the white group, suggesting the complex regulation of GH production and the limited effects of PSS-I on the inhibition of GH synthesis and somatic growth. The significantly increased NPY levels in the four LED groups (p < 0.05) indicated that these four LED spectra were effective in stimulating food intake and energy homeostasis. After two months, the gonads developed from chromatin nucleolar-stage oocytes to perinucleolar-stage oocytes in the four LED groups. The gene expression of Kiss2 and GPR54 in the four LED groups and of sbGnRH in the white and blue LED groups significantly increased when compared to that in the initial group (p < 0.05), while there were no significant differences in FSHβ and LHβ expression in the four LED groups. These results suggest that FSH and LH may not play important roles in gonadal development in juvenile red spotted grouper that are exposed to these four LED spectra.

Personality is defined as consistency in an individual's behavioral responses towards a given situation over time. Previous studies reported the presence of bold personality (BP) and shy personality (SP) in the olive flounder (Paralichthys olivaceus). Links between personality and the stress response have been studied in teleost fish; however, the response of SP and BP flounder to salinity stress is unknown. In this study, salinity tolerance of larvae at 25 days after hatch (DAH) and juvenile (50 DAH) flounders was investigated at salinities of 3, 5, 25, 45 and 50 parts per thousand (ppt). On 25 DAH, at salinities of 5 and 25 ppt, both SP and BP flounder keep 80% survival rate, while BP flounder larvae had higher survival rates at salinity of 45 ppt when compared with SP flounder. On 50 DAH, salinity tolerance of SP and BP flounder greatly improved, with 70% survival at salinitie of 3, 5 and 25 ppt but low survival rates (35%) at 50 ppt. At 45 ppt, BP exhibited slightly higher survival rate (80%) than SP flounder (60%) after a 96 h challenge. In addition, juvenile BP flounder (8 months) had lower plasma cortisol level, were more motivated to feed and showed more active responses during net confinement. Furthermore, Na+/K+/ATPase α1 (NKAα1) mRNA expression showed no significant difference between SP and BP flounder in SW, but was up-regulated after transfer to FW in SP and BP flounder. BP flounder expressed higher levels of gill Na+/K+/2Cl− cotransporter (NKCC1) mRNA than SP flounder in seawater (SW), while there were no differences between groups after transfer to freshwater (FW). Taken together, BP flounder had a low plasma cortisol level and an elevated appetite, as well as hyper-osmoregulatory ability. Selection for BP flounder in aquaculture is potentially important to improve animal welfare and increase production efficiency.

Urotensin I (UI), a member of the corticotropin-releasing hormone family of peptides, regulates a diverse array of physiological functions, including appetite regulation, defensive behavior and stress response. In this study, firstly, the tissue-specific distribution of UI mRNA in olive flounder (Paralichthys olivaceus) was characterized and we found that UI mRNA was highly expressed in caudal neurosecretory system (CNSS) tissue. Secondly, alignment analysis found that a conserved cAMP response binding (CREB) site and a TATA element were located in the proximal promoter of UI gene. In addition, treatment of forskolin activatated cAMP-CREB pathway and induced the up-regulation of UI mRNA in cultured CNSS, suggesting the role of CREB in regulating the UI mRNA expression. Furthermore, plasma UI concentration and UI mRNA in CNSS showed obvious daily rhythm, with higher values in the daytime while lower values in the nighttime. Thirdly, using bold personality (BP) and shy personality (SP) flounder as an animal model, we found that flounder exhibited significantly higher locomotor activity in the nighttime than in the daytime (P

In previous studies we employed multiple behavior assays, including propensity to feed, simulated trawl capture and escape response, to prove the presence of bold and shy personality (BP,SP) in olive flounder (Paralichthys olivaceus). However, the molecular mechanism of the different personality has not been elucidated. In this study, firstly, we found that the SP flounder had lower red blood cell count (RBC) and haemoglobin concentration (HBG) than BP flounder. Secondly, the transcriptomic profiles of the hindbrain in flounder with distinct personality were compared. A total of 144 differently expressed genes (DEGs) were identified, including 70 up-regulated and 74 down-regulated genes in SP flounder compared with BP flounder. Genes involved in hypoxia stress were detected in SP flounder, accompanied with down-regulation of ribosomal RNA synthesis. In addition, genes related with calcium signaling pathway, including endothelin, b-Fos, c-Fos and c-Jun were up-regulated in SP flounder. Furthermore, personality-related genes including UI, CCK, c-Fos showed significantly higher level in SP flounder than in BP flounder. GO enrichment analysis indicated that the GO categories "the tight junction pathway" and "lipid transport or localization pathway" were enriched in SP flounder, suggesting that the central nervous system homeostasis would be compromised. Thirdly, using a simple and scalable DNA methylation profiling method (MethylRAD), which allows for methylation analysis for DEGs in RNA-seq, we found that only part of gene expression was negatively associated with promoter methylation. Altogether, our study will not only lay a foundation for further studies on animal personality but also facilitate the selective breeding of olive flounder in aquaculture.

A neuromodulatory role for dopamine has been reported for magnocellular neuroendocrine cells in the mammalian hypothalamus. We examined its potential role as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder Paralichthys olivaceus . In vitro application of dopamine (DA) caused an increase in electrical activity (firing frequency, recorded extracellularly) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The dopamine precursor, levodopa (L-DOPA), also increased firing frequency, cell recruitment and enhanced bursting and tonic activity. The effect of dopamine was blocked by the D1, D5 receptor antagonist SCH23390, but not by the D2, D3, D4 receptor antagonist amisulpride. Transcriptome sequencing revealed that all DA receptors (D1, D2, D3, D4, and D5) were present in the flounder CNSS. However, quantitative RT-PCR revealed that D5 receptor mRNA expression was significantly increased in the CNSS following dopamine superfusion. These findings suggest that dopamine may modulate CNSS activity in vivo , and therefore neurosecretory output, through D5 receptors.

Whole-genome sequences of four EMS (ethyl methanesulfonate)-induced eggplant mutants were analyzed to identify genome-wide mutations. In total, 173.01 GB of paired-end reads were obtained for four EMS-induced mutants and (WT) wild type and 1,076,010 SNPs (single nucleotide polymorphisms) and 183,421 indels were identified. The most common mutation type was C/G to T/A transitions followed by A/T to G/C transitions. The mean densities were one SNP per 1.3 to 2.6 Mb. The effect of mutations on gene function was annotated and only 7.2% were determined to be deleterious. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed 10 and 11 genes, which were nonsynonymous mutation or frameshift deletion in 48-5 and L6-5 involved in the anthocyanin biosynthesis or flavone and flavonol biosynthesis. QRT-PCR results showed that only the Sme2.5_06210.1_g00004.1, which was annotated as UFGT (Flavonoid galactosidase transferase), expression significantly decreased in the L6-5 mutant compared with the WT. Also, the Sme2.5_06210.1_g00004.1 expression was lower in the colorless eggplant compared with colorful eggplant in the natural eggplant cultivar. These results suggest that Sme2.5_06210.1_g00004.1 may play a key role in eggplant anthocyanin synthesis.

Whole-genome sequences of four EMS (ethyl methanesulfonate)-induced eggplant mutants were analyzed to identify genome-wide mutations. In total, 173.01 GB of paired-end reads were obtained for four EMS-induced mutants and (WT) wild type and 1,076,010 SNPs (single nucleotide polymorphisms) and 183,421 indels were identified. The most common mutation type was C/G to T/A transitions followed by A/T to G/C transitions. The mean densities were one SNP per 1.3 to 2.6 Mb. The effect of mutations on gene function was annotated and only 7.2% were determined to be deleterious. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed 10 and 11 genes, which were nonsynonymous mutation or frameshift deletion in 48-5 and L6-5 involved in the anthocyanin biosynthesis or flavone and flavonol biosynthesis. QRT-PCR results showed that only the Sme2.5_06210.1_g00004.1, which was annotated as UFGT (Flavonoid galactosidase transferase), expression significantly decreased in the L6-5 mutant compared with the WT. Also, the Sme2.5_06210.1_g00004.1 expression was lower in the colorless eggplant compared with colorful eggplant in the natural eggplant cultivar. These results suggest that Sme2.5_06210.1_g00004.1 may play a key role in eggplant anthocyanin synthesis.

Corticotropin-releasing hormone (CRH) has been implicated in multiple physiological processes, such as circadian rhythms, food intake and anxiety-like behavior. In telelost fishes, CRH is predominantly expressed in the caudal neurosecretory system (CNSS), a much low level in hypothalamus and gonads, while undetectable levels in other tissues. However, the mechanisms governing this tissue-specific expression remain unknown. In this study, firstly, we investigated the expression pattern of CRH mRNA in different tissues of olive flounder (Paralichthys olivaceus). Secondly, we found that flounder exhibited low locomotor activity in the daytime, concomitant with the highest CRH mRNA expression in CNSS at noon. Thirdly, we examined whether epigenetic mechanisms through DNA methylation are involved in tissue-specific expression of CRH mRNA. Promoter methylation of the CRH gene was assessed through bisulphate sequencing methods. In the proximal promoter, almost no methylation was detected in the muscle, hypothamus and CNSS. However, different methylation was detected in the distal promoter of CRH. The methylation was 63% in CNSS, while that in hypothalamus and muscle was 85% and 96%, respectively. Lastly, bioinformatics analysis revealed that a conserved AP-1 binding site (5-TCACTGA-3) was located in the proximal promoter of CRH gene. In vivo experiment, lipopolysaccharide (LPS) intraperitoneally injection in the flounder up-regulated c-Fos and CRH mRNA in CNSS (P 0.01). In CNSS tissue culture experiment, forskolin treatment significantly induced the expression of c-Fos, c-Jun and CRH mRNA (P 0.01). Collectively, our data provide the evidence that distal promoter methylation and c-Fos signal pathway are involved in transcriptional regulation of CRH expression in flounder.

The peptide urotensin II (UII) mediates multiple physiology effects in mammals and fishes, and UII expression shows a tissue-specific pattern. However the mechanism is still unknown. In the present study high level of UII mRNA was detected in the caudal neurosecretory system (CNSS) of the olive flounder when compared to other tissues. We examined whether epigenetic mechanisms of DNA methylation are involved in UII gene expression. Methylation DNA immune precipitation (MeDIP) assay showed low methylation of UII promoter in CNSS tissue compared with muscle and spinal cord. Methylation of UII promoter was further assessed through bisulphate sequencing analysis. Low level methylation (31%) in CpG island of UII promoter was detected in CNSS tissue, while methylation status in muscle and spinal cord was 89% and 91%, respectively. In addition, high conserved sites of Hoxd4 in UII promoter were found. Activation of Hoxd4 mRNA using transretinoic acid (RA) resulted in 18-fold increase of UII mRNA expression in CNSS and high locomotor activity in medaka, confirming that Hoxd4 is also involved in UII gene transcriptional regulation. Taken together, our data provide the first evidence of the epigenetic mechanism of promoter methylation in transcriptional regulation of UII expression in a tissue-specific manner, and Hoxd4 may also participate in UII gene transcription in flounder.

Diploid and triploid hybrids of large yellow croaker (Pseudosciaena crocea, Pc) and Miiuy croaker (Miichthys miiuy, Mm) were produced by interspecific hybridization. The result showed that Pc (♀) and Mm (♂) can be successfully crossed and hybrid offspring of normal hatch rate were produced. In contrast, a lower fertilize and hatch rate were produced for hybrids of Pc (♂) and Mm (♀), and the offspring died day 19 after hatching. To confirm the ploidy of hybrid offspring, we examined the karyotypes of offspring. The result of DNA relative content investigation and genetic relationship analysis demonstrated that the major hybrid offspring were hybrid diploid. In addition, a small number of hybrid were triploid and gynogenesis diploid. Morphological analysis based on the measurable traits data indicated the success of the interspecific crosses. The natural gynogenetic diploid large yellow croaker (PP), diploid hybrids (PM) and triploid hybrids (PPM) were closer to maternal parent (Pc) than paternal parent (Mm). This study provided genetic evidences of the chromosome, DNA content and morphological levels to support the successful establishment of the polyploidy hybrids of Pc (♀) and Mm (♂).

Glucocorticoid receptor (GR) is transcribed in a tissue- and cell-specific manner with multiple exon 1 mRNA variants driven by selective promoters. We recently cloned and characterized the 5.3kb proximal promoter sequence of porcine GR gene containing 7 untranslated alternative first exons each processed by a distinct promoter. In this study, we showed tissue-specific expression of total GR and its exon 1 mRNA variants in hippocampus, muscle and liver of pigs. Total GR mRNA was most abundant in liver, followed by muscle and hippocampus in descending order. Among all the GR exon 1 mRNA variants detected, GR exon 1-9/10 and 1-4 were the most predominant variants in all the three tissues. The abundance of GR exon 1-4 mRNA was similar to that of 1-10 in muscle, but was significantly lower than 1-10 in liver and hippocampus. The activities of truncated short (S) and long (L) promoters of respective GR exon 1 mRNA variants were analyzed by luciferase reporter assay in 3 representative cell lines, SY5Y, C2C12 and HepG2. S1-10 and S1-4 demonstrated significantly higher activities than other short promoters in all the cell lines examined. Nevertheless, the strongest activity and cell specificity were detected for L1-10 promoter, which was consistent with the predominant exon 1-9/10 expression in porcine tissues. Moreover, with 3 potential nGRE binding sites, L1-10 promoter was more sensitive to dexamethasone (DEX) in HepG2. Our data provide basic knowledge of the transcriptional mechanism underlying the tissue- and cell-specific expression of porcine GR under basal or ligand-stimulated conditions.

Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (P

In the present study the influence of long photoperiod (LP, 16L:8D) and short photoperiod (SP, 8L:16D) on hepatic energy metabolism in the olive flounder (Paralichthys olivaceus) was investigated. Flounders were maintained under LP or SP conditions for 2 weeks then plasmatic and hepatic parameters were assessed. At the plasmatic level, the concentration of cortisol was enhanced in flounder maintained under LP compared to SP. Alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities in plasma also increased in LP flounder. There was no significant difference in plasma glucose levels between the two experimental groups. Plasma osmotic pressure, Na and Cl levels were significantly higher in LP compared to the SP group. In liver, a significant decrease of triglycerides together with an increase in glycogen was observed in the LP group. Hepatic hsl and pepck and muscle hsl mRNA expression in LP was significantly higher in the SP group. Overall the results indicate that the LP treatment caused a mild stress response and increased hepatic energy metabolism in the flounder, which in turn could affect osmoregulation. In conclusion, it would appear that LP treatment can adversely influence hepatic energy metabolism in adult olive flounder under fasting condition.

MicroRNA (miRNA) biogenesis is determined mainly by Drosha, Dicer and Argonaute2 (Ago2). Different breeds of pigs with vast differences in serum cortisol level demonstrate distinct profiles of hepatic miRNA expression. As yet, little is known about whether glucocorticoid contributes to the breed differences in miRNA biogenesis. Here, we used newborn Large White (LW) and Erhualian (EHL) piglets to investigate the role of glucocorticoid in breed-specific hepatic miRNA biogenesis. Erhualian piglets showing significantly higher serum cortisol level, as compared to LW, demonstrated higher hepatic expression of Drosha, Dicer and Ago2 at the protein level, but not at the mRNA level. At the post-transcriptional level, miRNAs that are predicted to target these proteins may be involved in the regulation. Hepatic expression of miR-15b and miR-222 was significantly lower in EHL piglets and was associated with higher glucocorticoid receptor binding to the respective promoter regions of miR-15b and miR-222 genes. The inhibitory effect of glucocorticoid on miR-15b and miR-222 expression was further verified in HepG2 cells, in which dexamethasone significantly downregulated the expression of primary transcripts of miR-15b and miR-222 genes. In conclusion, the higher protein content of Drosha, Dicer and Ago2 in the liver of EHL piglets is post-transcriptionally regulated, at least in part, by glucocorticoid-mediated repression of miR-15b and miR-222.

Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P

A mechanism of survivability recovery, whereby component is the basic unit of being fixed, is presented for information system in this paper in order to overcome the deficiencies existed in approach on survivability recovery. Services provided in information system are classified into complete service, acceptable service and interrupted service by the degree of dependence on disable failed critical component. The survivability of information system is measured by availability of component. Procedure of survivability recovery can be divided into two phases in time domain. The first phase is to restore interrupted service by fixing the disabled critical components. Acceptable service can be perfected by fixing the disabled non-critical components in the second phase. The experiment is carried out by applying the mechanism mentioned above in a networked teaching system. The whole process of survivability recovery is demonstrated in detail. The results shows that it is effective to determine recovery actions and activating sequence, increase the capability of recovering rapidly and diversify methods of recovering survivability in information system. The work done by author has a major guiding significance in analyzing and recovering survivability for actual information systems.

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P

Background Mitochondria, which are essential for the functionality of eukaryotic cells, are particularly important in metabolically active tissues such as liver. Different breeds of pigs demonstrate distinct metabolic profiles in the liver, yet little is known whether the expression and transcriptional regulation of mitochondrial genes differ between breeds. Results Here we used male newborn Large White (LW) and Erhualian (EHL) piglets to delineate the difference in hepatic mitochondrial gene regulation between breeds. The hepatic content of ATP was significantly higher (p IDH2) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit d (ATP5H) were expressed significantly lower, while adenylate kinase 1 (AK1) was significantly over expressed in EHL piglets. Compared to LW, the over expression of mtDNA-encoded genes in EHL was associated with significantly higher (p AK1 and ATP5H. Moreover, higher expression of AK1 in EHL piglets was also associated with lower cytosine methylation (p Conclusions These results indicate a role of the GR in the breed-dependent regulation of mitochondrial genes in the liver of newborn piglets.