Roles of plasmid-encoded proteins, EseH, EseI and EscD in invasion, replication and virulence of Edwardsiella ictaluri.

Native plasmids pEI1 and pEI2 were detected in Edwardsiella ictaluri HSN-1 isolated from diseased yellow catfish (Pelteobagrus fulvidraco). EseH encoded by pEI1 and other two proteins, EseI and EscD, encoded by pEI2, were found with homology to type III secretion system (T3SS) proteins. To investigate their roles in pathogenesis, the native plasmids were cured based on plasmid incompatibility by introducing a Kan positive and SacB negative selection marker into gene spacer of the native plasmids. Mutants with the deletion of the target genes were obtained by reverse PCR and self-ligation, and all mutants were examined for their virulence effect in yellow catfish. Compared with the HSN-1 strain, the two mutants ΔeseH and ΔeseI were attenuated, while mutant ΔescD had increased virulence with higher Competitive Index (CI) value. The adherence and invasion assays on fish EPC cells indicated that ΔeseH and ΔeseI had decreased ability in adherence. Using E. tarda as surrogate, EseH and EseI were detected in culture supernatants, but EscD was not, with the secretion of EseH depending on T3SS. In addition, EseH and EseI were found translocated into host cells, and by means of subcellular fractionation, EseH was localized in membrane fraction of ZF4 cells, and EseI in the cytosol fraction. Hence, the role of these three genes in adherence, invasion and cellular replication was revealed from the pathogenic bacterium E. ictaluri.

Molecular cloning, tissue distribution, and ontogenetic expression of ghrelin and regulation of expression by fasting and refeeding in the grass carp (Ctenopharyngodon idellus).

Ghrelin is a recently discovered brain-gut peptide in fish and mammals. It has two main physiological functions-stimulating growth hormone secretion and regulating appetite. To investigate the biological function of ghrelin in appetite regulation in grass carp Ctenopharyngodon idellus, the full-length cDNA sequence of the ghrelin gene was obtained by RT-PCR and rapid amplification of cDNA ends methods. Homology analysis indicated that ghrelin cDNA sequence is conserved in teleosts. The grass carp ghrelin gene consists of four exons and three introns, which is similar to that of common carp, zebrafish and humans. Real-time quantitative PCR was used to detect ghrelin mRNA expression in the study. In adult tissues, high levels of gene expression were found in the foregut; moderate levels in the muscle, liver, hypothalamus, white adipose tissue, midgut, heart, and pituitary; and lower levels in the three other examined tissues. During embryonic development, ghrelin mRNA expression could be detected as early as fertilized egg stage and displayed an increase in expression until heart appearance stage. After hatching, the level of ghrelin mRNA expression decreased sharply to the lowest level at 1 day post-hatching (dph), then increased to a higher level at 7 dph and subsequently decreased to a relative stabilized level from 15 to 35 dph. The ghrelin mRNA expression was significantly upregulated in the brain and intestine during fasting and was downregulated after refeeding. The results suggest that ghrelin mRNA expression has obvious tissue specificity and may have a role in appetite regulation in grass carp.

Purification and characterization of 6-phosphogluconate dehydrogenase (6-PGD) from grass carp (Ctenopharyngodon idella) hepatopancreas.

6-Phosphogluconate dehydrogenase (6-PGD, E.C.: 1.1.1.44) was purified and characterized from the hepatopancreas of grass carp (Ctenopharyngodon idella) for the first time. Grass carp represents the second largest aquaculture industry in the world after silver carp, constituting 14.7% of the world aquaculture production, with an average annual increase of 14% in China, mainly as a source of food. The purification procedure involved a single 2', 5'-ADP-Sepharose 4B affinity chromatographic step by using different elution buffers. The enzyme was purified 309-fold with a specific activity of 5.259 U/mg protein and yield of 68%. The purity and subunit molecular weights of the 6-PGD were checked on SDS-PAGE and purified enzyme showed a single band on the gel. The subunit molecular mass was 57 kDa, with an optimum pH, temperature and ionic strength at 7.96, 50 degrees C and 100 mM Tris-HCl, respectively. The Km values of 6-PGA and NADP+ were 0.019 and 0.0052 mM, respectively, while Vm of 6-PGA and NADP+ was 0.69 U/ml. Dissociation constants (Ki) for 6-PGA and NADP+ were 2.05 and 0.12 mM, respectively. NADPH inhibited the enzyme in a competitive manner and its Ki value was 0.032 mM. The Cu2+, Zn2+, Cd2+ and Al3+ showed inhibitory effects on the enzyme with IC50 values of 0.293, 0.099, 0.045 and 1.526 mM, respectively. All tested metals inhibited the enzyme in a competitive manner, indicating that these metals might be toxic even at low concentrations for the 6-PGD. As the fish is one of valuable foodstuff of animal sources for human consumption, under certain environmental conditions, metal ions accumulated in fish up to a lethal concentration may be harmful for human health. Therefore, it is impending to reduce the concentration of metal ions in contaminated lakes and rivers for fishery and also for human health.

Molecular characterization of cholecystokinin in grass carp (Ctenopharyngodon idellus): cloning, localization, developmental profile, and effect of fasting and refeeding on expression in the brain and intestine.

Cholecystokinin (CCK) is a multi-functional brain-gut peptide in fish and mammals. To investigate the role of CCK in appetite regulation in fish, a 770-bp full-length cDNA sequence of CCK gene was obtained by RT-PCR and rapid amplification of cDNA ends methods in grass carp Ctenopharyngodon idellus. Homology analysis showed that the CCK cDNA sequence of grass carp had the highest similarity (90 %) to that of goldfish Carassius auratus and a higher similarity (>70 %) to those of other teleosts than to mammals. The PCR amplification using genomic DNA identified that the CCK gene of grass carp was comprised of three exons and two introns. Real-time quantitative PCR was used to detect CCK mRNA expression in adult tissues. High levels of gene expression were found in the hypothalamus and pituitary; moderate levels in the intestine, muscle and white adipose tissue; and low levels in other tissues. During early development (i.e., fertilized eggs to 35-day post-hatching larvae) the levels of CCK mRNA expression were higher during embryonic developmental stages than during post-hatch larval stages. Fasting decreased CCK mRNA expression levels in the brain and intestine, whereas refeeding resulted in an increase of expression. The results suggest that CCK mRNA expression has obvious tissue specificity and may have a role in feed intake regulation in grass carp.

[Individual fecundity of Culter alburnus in Xujiahe Reservoir of China].

In April - June 2008, the matured female Culter alburnus specimens were collected from the Xujiahe Reservoir of China. Their ages were identified by the scales, and their body length, body mass, gonad mass, and other morphological indices were measured. Counted with gravimetric method, the individual absolute fecundity was obtained, and used for calculating the relative fecundity per centimetre, relative fecundity per gram, maturity, relative fatness, and other indices. Based on these measurements and calculations, the relationships between the individual fecundity and the morphological indices of C. alburnus in the Reservoir were studied. The ages of the specimens ranged from 2+ to 5+ years old, and the individual absolute fecundity and relative fecundity per centimetre were exponentially correlated with body length and body mass, and linearly correlated with age. No significant correlations were observed between the relative fecundity per gram and these morphological indices. Multi-regressive analyses showed that the individual absolute fecundity was significantly correlated with body mass and gonad mass, the relative fecundity per centimetre was significantly correlated with gonad mass, relative fatness and maturity, while the relative fecundity per gram was only correlated with maturity.

[Seasonal dynamics of phosphorus forms in water body and sediments of Nanhu Lake, Wuhan].

Water samples and sediment cores were taken at 5 sites of Nanhu Lake, Wuhan to investigate the seasonal dynamics of phosphorus in water body, sediments and their interstitial water, as well as the phosphorus forms in sediments. The results showed that the average concentrations of total phosphorus (TP) and orthophosphate (PO4(3-) -P) in water body were 0.240 and 0.033 mg x L(-1), respectively, with the highest concentrations of TP and PO4(3-)-P in November and the lowest concentration of TP and of PO4(3-) -P in February and August, respectively. The concentration of TP in sediments was averagely 1.005 mg x g(-1), being increased from April to November and becoming stable from November to February, and decreased with sedimentation depth. The concentration of PO4(3-) -P in interstitial water was 0.209 mg x L(-1) on average, being the highest in November and the lowest in February, with the same vertical distribution pattern like TP in sediments. The dominant form of phosphorus in sediments was inorganic phosphorus (IP), occupying 61.4%-77.1% of total extracted phosphorus. The concentration of bio-available phosphorus (BA-P) in sediments was very high, but decreased significantly with sedimentation depth. According to the significant correlations between phosphorus forms in sediments and PO4(3-) -P in interstitial water, the BA-P and auto-calcium bound phosphorus (ACa-P) in sediments should be the main P sources released from sediments to water body.