Abstract:In order to provide basic data for the oncoming bio-manipulation based on fish and reference system for hereafter investigating its subsequent effect on fish composition in a newly established water supply reservoir (Qingcaosha Reservoir in Shanghai of China), fish assemblages in this reservoir were sampled by multimesh gillnets from Jul. to Oct. in 2010. A total of 993 individuals, belonging to 18 species, representatives of 5 orders, were caught, of which Cypriniformes accounted for 72.2% of the total species number. Based on importance value, Hemiculter bleekeri, Coilia nasus, Carassius auratus and Pseudobagrus nitidus were the dominant species. Clustering analysis (CA) showed that fish assemblages significantly differed spatially, and there were marked effects of net size on species composition of fish catch. One-way ANOVA indicated that there were no significant spatial differences in fish diversities. Although Shannon’s species diverisity (H^’) was conspicuously positively correlated with Margalef’s species richness (R) and evenness (J), respectively, H^’was governed more by R. Invertivorous (44.4%) and omnivorous (33.3%) fishes prevailed in species number among the 4 feeding functional groups, and sedentary fishes (77.8%) predominated among the 4 ecological groups. Contrasting to the adjacent estuarine zone with more species of Perciformes and dominated by estuarine and/or inshore fishes, Qingcaosha Reservoir was characterized by dominance of freshwater or low salinity water fishes. It is presumed that isolation and decreased salinity that resulted from reservoir construction would be responsible for the fish characteristics of Qingcaosha Reservoir.

Abstract:The rock bream, Oplegnathus fasciatus, a subtropical and carnivorous species, is an economically important marine fish in East Asia. The high commercial and ornamental value makes it a promising aquaculture species in the future. However, to some extent, the lack of information on ontogenetic development has restricted the breeding industry of this species. In this study, the allometric growth in rock bream was analyzed. The results are as follows: at the general condition for fingerling-production, the total length and body weight of rock bream larvae were measured from hatching to 50 days post hatching. The increase of total length and body weight could be estimated with the Cubic function and took on the S-Curve. The curve could be divided into three phases and each phase possessed different growth rate. The head length, head height, trunk height, eye diameter, mouth width, rostrum length, abdomen length, and tail fin length of rock bream were measured and the relationship between them and the total length was analyzed. The result showed the allometric growth in rock bream larvae. By analyzing the inflexion points in growth curves, in combination with morphological development of the larvae, we found that some important organs (head, mouth, eye, digestive tract and fins) had developed prior to other ones. In rearing rock bream larvae, the best environmental condition should be established by making the important organs prior development.

Abstract:Serum biochemical parameters were investigated in juvenile Epinephelus septemfasciatus (300 ± 20 g) subjected to low-temperature stress. The experimental fish at a temperature of 12.4 ℃ were directly shifted to a lower temperature seawater (8.0 ℃) for 0, 1, 2, 5, 10 days. The results showed that, the activity of serum AKP showed an irregular variation trend as a result of low temperature stress, and was significantly higher than that in cold water at 2 days. Enzyme activity of GGT showed a parabolic trend with the maximum efficiency in cold water for 2 days, and minimum for 10days. Glutamate-oxaloacetate (GOT) and glutamyl-transpeptidase (GPT) were increased with the increasing stress duration, and the activities of GOT and GPT at low temperature were significantly higher than those in control group. Enzyme activities of LDH showed insignificant change during the experimental duration. The concentration of serum total protein (TP) showed a descending trend. There was a slight change in concentration of GLU during the prophase of experiment, while the concentration of GLU exposed to low temperature stress at 10 days was 3.9 times high than that in the control group. The concentration of serum TG decreased at earlier stage and mounted at the end of experiment, and no significant difference in TG concentration was found between the initial and end of experiment. CREA concentration showed a similar trend with the TP concentration, declined to 22% of original concentration (48.57±34.96 mmol/L). During the whole experimental duration, no significant difference was found in serum ionic density. In conclusion, juvenile fish with similar size in this experiment can withstand sudden low-temperature change stress and the stress has cumulate effects on juvenile Epinephelus septemfasciatus, indicating that during winter the time of fish kept at 8 ℃ should not exceed 10 days in practice.

Abstract:The scalloped hammerhead shark (Sphyrna lewini) is circumglobally distributed along continental margins and oceanic islands in tropical waters. With the development of fishery resource, S. lewini is one of the most affected and ecologically important species. It is very important to investigate the genetic variance and population structure of this species. However, there are no genetic investigations for this fish in China. In the present study, thirty-four individuals were collected to analyze the genetic diversity and genetic structure for this species. A 548 base pair (bp) fragment of the hypervariable portion of the mtDNA control region was sequenced and sequences were edited and aligned. The fragments revealed 11 polymorphic sites, which defined 5 haplotypes. The results of present study showed that haplotype diversity, nucleotide diversity and mean number of pairwise differences of Rizhao population (h=0.6000±0.1305; π=0.0046±0.0031; p=2.5333±1.4856) were a little higher than those of Xiapu population (h=0.5109± 0.0955; π=0.0024±0.0017; p=1.2899±0.8373). A Neighbour-Joining (NJ) tree of the control region haplotypes was constructed based on the outgroup S. zygaena and two haplotypes from East Pacific which were downloaded from NCBI. Two haplotypes from East Pacific were clustered with three haplotypes from the present study and two lineages were distinguished by 1.6% sequence divergence. The pairwise fixation index F_st=－0.047 (P=0.914) revealed no significant genetic differentiation between these two populations. The exact test of population differentiation (non-differentiation exact P values) following sequential Boferroni correction showed that no significant differences (P<0.01) existed among between two populations, which supported null hypothesis. The mismatch distribution analysis of all the S. lewini sequences was bimodal, and one small mode corresponded to internal clade comparisons and the other to differences between individuals among clades. Low levels of genetic diversity in Chinese Coast Sea for S. lewini may be a consequence of small effective population size which restricted gene flow. The results of present study provided a deeper understanding of the processes that led to existing levels and patterns of genetic diversity.

Abstract:The lipid classes of Antarctic krill meal were analyzed, and the fatty acid compositions of different lipid components were comparatively studied by gas chromatography/mass spectrometry (GC/MS) after preparation by thin layer chromatography and acid-catalyzed esterification. The main results were as follows: ①The lipid components were abundant in Antarctic krill meal, and the content of total lipids was up to 11.37%. The total lipids were primarily composed of triglycerides, phospholipids and free fatty acids, while the total cholesterol content was low. ②The predominant fatty acids of total lipids were C16:0, C18:1n-9, C18:1n-7, C20:5n-3 and C22:6n-3, and the total content of C20:5n-3 and C22:6n-3 reached 30.67%, which indicated that Antarctic krill meal had the high nutritional value and great potential for development in lipids. ③Significant differences were demonstrated among the fatty acid compositions of different lipid components. The saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) of cholesterol esters and triglycerides were significantly higher than those of free fatty acids and phospholipids (P<0.05); whereas the polyunsaturated fatty acids (PUFA) of free fatty acids and phospholipids were 48.50% and 49.96%, respectively, and were markedly more than 13.11% in cholesterol esters and 24.36% in triglycerides (P<0.05).

Abstract:Using fluorescence in situ hybridization (FISH), 5S rDNA gene was mapped on chromosomes of half-smooth tongue-sole(Cynoglossus semilaevis)and olive flounder(Paralichthys olivaceus)for the first time. The results showed that 5S rDNA has two hybridization signal loci in a pair of homologous chromosomes of male and female half-smooth tongue-sole, respectively; 5S rDNA has two and three hybridization signal loci in a pair of homologous chromosomes of diploid and triploid olive flounder, respectively, referring to the clear and specific hybridization signals. FISH mapping of 5S rDNA genes on the chromosomes of diploid half-smooth tongue-sole and triploid olive flounder provides a feasible method for ploidy and chromosome identification of half-smooth tongue-sole and olive flounder. In addition, the coding region sequences of 5S rDNA gene of turbot (Scophthalmus maximus) and half-smooth tongue-sole were amplified using same primers designed for olive flounder 5 S rDNA gene, and the phylogenetic analysis of the 5S rDNA genes in four species of flatfishes, including plaice(Pleuronectes platessa), senegalese sole (Solea senegalensis), largemouth bass(Micropterus salmoides) and arctic lamprey(Lampetra japonica) showed that the 5S rDNA gene of five species of flatfishes shared high homology (98.3%) and gathered into a branch. The GC content was significantly higher than the AT content in conservative sequence of 5S rDNA coding region of these five species of flatfishes.

Abstract:Effect of starvation on gastric evacuation and associated mathematical models in juvenile southern catfish (Silurus meridionalis)

Abstract:The major histocompatibility complex (MHC) class II A gene plays an important role in the immune response of teleost. In this study, the full length of MHC IIA cDNA and genomic sequences were obtained from healthy Oreochromis niloticus by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length of cDNA sequence (Orni-DBA*0101, GenBank No: JF719813) comprises 1 205 bp with a 720 bp open reading frame (ORF) encoding a predicted protein with 239 amino acid residues. The genomic sequence was further identified to be 1 388 bp in length, which contains four exons and three introns. Eight class IIA alleles were identified from four healthy O. niloticus individuals. The variability that existed in the α-1 region was higher than that of half-smooth tongue sole. All the characteristic domains present in MHC IIA of other species could be found in O. niloticus MHC IIA sequence, including a leader peptide, two extracellular domains, a transmembrane region, and a cytoplasmic domain. In addition, there are four conserved cysteine residues and abundant phosphorylation sites. The deduced amino acid sequence shares 23%－65% identity with those of other vertebrates. Semi-quantitative reverse transcriptase PCR (RT-PCR) demonstrated that O. niloticus MHC IIA mRNA was ubiquitously expressed in all tested tissues, which is highly expressed in spleen, kidney, gills, liver, heart, gonad and gut, however, lowly expressed in muscle and bladder. Challenge of O. niloticus with pathogenic bacteria, Aeromonas hydrophila, results in a significant change of the expression of MHC IIA in the liver, spleen, kidney, gills and intestine. The result implied that MHC IIA might play an important role in the immune system.

Abstract:An 88-day feeding test was conducted to estimate the effects of dietary lipid to protein ratios on growth performance, body composition and non-specific immunity in largemouth bass with initial body weight (10.06±0.02) g. Eight diets(D1-D8)were formulated with varying lipid to protein (LIP/PRO, w/w) ratios ranging from 0.17 to 0.84. From D1 to D8, dietary lipid levels were increased (9.0%-26.5%), while dietary protein levels were decreased (52.0%-31.0%). The results of this study suggested that the specific growth rate, feed efficiency ratio (FER), dietary protein digestibility coefficient, dietary lipid digestibility coefficient and lipid deposition of D2-fed fish were the highest. The protein efficiency ratio was significantly higher in fish fed D3-D5, compared with fish fed D1 and D8. The protein deposition rates of D2- and D3-fed fish were significantly higher than those in fish fed D1 and D5-D8. The dietary energy digestibility coefficients among the fish fed D1-D4 were significantly higher than those with other treatments. Excessive dietary lipid had a significant effect on body composition with increased body lipid deposition. The survival rates were significantly higher in fish fed D1 and D2 with higher protein levels, compared with those in fish fed D5-D8 with lower protein levels. The highest activity of lysozyme, CH50 and respiratory burst of head kidney leukocytes occurred in D4-, D2- and D2-fed fish, respectively. According to a one-way ANOVA against dietary treatment, the optimal dietary protein and lipid levels for growth and FER of largemouth bass are 49.30% and 11.50%, respectively. Using a second-order polynomial regression analysis, the optimum dietary protein, lipid and lipid to protein ratio (L/P) in the diet for largemouth bass are estimated, based on the specific growth rate (SGR) and protein deposition rate (PDR). With the highest SGR, the optimum dietary protein, lipid and L/P are estimated to be 48.20%, 12.44% and 0.26. With the highest PDR, the optimum dietary protein, lipid and L/P are estimated to be 46.42%, 13.96% and 0.30. A quadratic regression analysis of PDR against dietary protein to energy ratio (P/E) indicates a similar result that the optimum dietary P/E, protein, lipid and L/P requirements of largemouth bass are 23.72 mg/KJ, 46.16%, 14.18% and 0.31, respectively. Based on those results, it could be concluded that largemouth bass growth performance, body composition and non-specific immunity are affected by dietary lipid and protein levels to different extents, and that an excess of dietary lipid inhibits digestion of dietary protein, suggesting that protein-sparing effect of dietary lipid is limited in this fish species. It could be recommended to maintain dietary protein and lipid levels within 46%-49%, and 11.5%-14%, respectively, in the practical feed for largemouth bass.

Abstract:Dmrt family is one of the oldest developmental gene family, and is important to the embryo organizes normal growth, Spermatogenesisspermatogenesis, and the nervous system and sense organ development. Dmrt2 is one of the members of Dmrt family, were which was cloned in mammals such as human, birds such as Gallus gallus, Aquatic aquatic animals such as Oryzias latipes, are is closely related to gonad Development development and growth. RACE -PCR technique wasere used to clone Dmrt2 gene in Andrias davidianus, 2 026 bp full-length cDNA was obtained, containing 1 581 bp open reading frame(ORF), 58 bp 5′’UTR, 387 bp 3′’UTR, GenBank number is FJ859987. This sequence encoded 526 amino acids, and the amino acid sequence was across the membrane protein, no signal peptide, and the protein positioning in the cytoplasm. The amino acid sequence was analyzed by the Conserved Domain Database(CDD), surveyed that single amino-terminal DM domain is from 93 bp to 146 bp, contains a pattern of conserved zinc chelating residues C2H2C4, super-family pfam00751 and smart003010. Blast DM domain with Homo sapiens mab-3, Gallus G. gallus Dmrt2, Xenopus (Silurana) tropicalis Dmrt2 and Danio rerio mab-3, five amino acid were had variation, include Ile, Arg, Met, Thr, Cys. Phylogenetic analysis indicated that the five species above of Homo sapiens , Gallus G. gallus , Xenopus X. (Silurana) tropicalis and Danio D. rerio were clustered first, that means Dmrt family is highly conserved during evolution, and the five amino acids variation have had little affection effect onto protein functions. RT-PCR was used to detecte detect the expression of Dmrt2 in skin, intestines, gills, muscle, stomach, spermary and kidney of Andrias A. davidianus, it and results showed it is high level expression only in spermary and muscle, which is indicated that the Dmrt2 gene maybe play an important role in gonad development and growth development.

Abstract:Perkinsus olseni(P. olseni) is one of the important pathogenic parasites of the shellfish. With the purpose of building a rapid, sensitive, accurate and easy to use detection method for P. olseni, we established a P. olseni loop-mediated isothermal amplification assay(LAMP) based on the internal transcribed spacer(ITS) of Perkinsus olseni 5.8S rDNA sequences. We used the online software Primer Explorer V4 (http://primerexplorer.jp/e/) and designed a set of 4 LAMP primers(Perk-FIP, Perk-BIP, Perk-F3 and Perk-B3) of the P. olseni, then we optimized the reaction conditions, mainly about the reaction temperature, magnesium ion concentration of the reaction system and the reaction time. After that, we got the P. olseni 25 μL LAMP reaction system, including: 2.5 μL 10′ThermoPol Reaction Buffer, 4 μL dNTPs(10 mmol/L each), 5 μL Betaine(5 mol/L), 1.6 μL Perk-FIP(25 μmol/L), 1.6 μL Perk-BIP(25 μmol/L), 1 μL Perk-B3(5 μmol/L), 1 μL Perk-F3(5 μmol/L), 4 μL MgCl2(25 mmol/L), 2.3 μL sterile water, 1 μL Bst DNA polymerse(8 000 U/mL) and 1 μL DNA template, the optimal reaction temperature is 64 ℃ and the optimal reaction time is 60 min. In this research, the LAMP products were detected mainly using agarose gel electrophoresis and visual inspection of a color change due to addition of ?uorescent dye. Before confirming the minimum threshold of the LAMP, we constructed P. olseni positive plasmid, also based on P. olseni 5.8S rDNA ITS sequences. The result shows that the minimum threshold of the LAMP assay is approximately 30 copies of plasmid DNA. We proved that the developed LAMP method was highly specific for P. olseni, and no cross-reaction was observed with other pathogens, such as Perkinsus marinus(P. marinus) , Bonamia exitiosa(B. exitiosa) , Ichthyobodo sp. and Acute Viral Necrosis Virus(AVNV). A comparative evaluation of the LAMP and PCR assays using 20 Ruditapes philippinarum(R. philippinarum) samples showed that LAMP is more sensitive and accurate than PCR and the shellfish parasite P. olseni is widely distributed in farming shellfish of North shellfish farming area. Totally, these results indicate that the LAMP method is a kind of simple, sensitive, specific, and reliable technique for the detection of P. olseni. The LAMP technique could be used for the detection of P. olseni in the coastal shellfish farms and laboratories with simple equipment.

Abstract:The ribosomal DNA internal transcribed spacer 1 (ITS1) of three wild populations of Exopalaemon carinicauda was cloned and sequenced. The phylogenetic relationship among Palaemoninae was discussed using ITS1 from GenBank database. The results show that the length of the ITS1 ranged from 345 to 384 bp. The mean contents of GC were higher than AT;79 variation sites and 39 haplotypes were obtained in ITS1; Nucleotide diversity and average number of nucleotide diferences were calculated to analyze the gene sequence variation. Eight kinds of microsatellites were found in E. carinicauda. The type of (GA) _n was rich in ITS1. AMOVA analysis results show that the genetic differentiation of the three groups was weak or only moderately differentiated.The molecular phylogenetic tree was constructed with NJ method using software MEGA 4.0 to study several species of the Palaemoninae molecular phylogeny. According to the NJ tree, different individuals of the same species, different species of the same genera were grouped together, in consistence with the morphological taxonomy.

Abstract: Sebastes schlegelii is an important economic fish cultured in North China. In recent years, S. schlegelii aquaculture has developed rapidly along the northern coast of China. The expansion and intensification of S.schlegelii farming has led to the occurrence of some diseases. In August 2011, an epizootic occurred among cultured S.schlegelii in a cage culture farm in Qingdao, which caused cumulative mortality rate 30%. To make certain the causative pathogen, we studied the etiology and virulence of S. schlegelii so as to provide basic data for the farming industry of S. schlegelii. In this study, we used the morphological, physiological and biochemical methods to identify the isolated bacterium. Meanwhile, to ensure the results accurately, the phylogenetic analyses were also used. A dominant bacterium strain SS-1 was isolated from the skin, gill, liver, spleen, kidney of the diseased S. schlegelii. Artificial infection test with intramuscular injection method proved that the median lethal concentration (LC50) value of bacterium strain SS-1 in S. schlegelii was 9.6×106 CFU/mL. This bacterium could cause severe infections in several tissues and organs of the fish. The results of morphological, physiological & biochemical characteristics tests showed that strain SS-1 was gran-negative, rod bacteria, polar flagellum, and the results of oxidase, contact enzyme, nitrate reduction, producing indol, V-P test and arginine digydrolase test were positive; producing H2S test was negative. The morphological, physiological & biochemical characteristics indicated that SS-1 could be identified as Listonella anguillarum. The sequence analysis of 16S rRNA gene of strain SS-1 was identical with L. anguillarum and the homogeneity is 99%. This paper reported for the first time that L. anguillarum caused the disease of S. schlegelii in China, which will provide reference in fish health management and disease control.

Abstract:Full-length cDNA sequence of 14-3-3 gene was isolated from the eyestalk of mud crab Scylla paramamosain through RT-PCR and RACE. Sequence analysis indicated that 14-3-3 gene had an open reading frame of 744 bp encoding 247aa of 28.086 ku and pI at 4.675. The amino acid sequences of 14-3-3 gene possessed 95%, 93%, 92% identity with the 14-3-3 genes of Penaeus monodon, Fenneropenaeus merguiensis, Megachile rotundata respectively. 14-3-3 protein firstly clustered with 14-3-3 proteins of Penaeus monodon and Fenneropenaeus merguiensis in the phylogenetic analysis. The expression of 14-3-3 gene in tissues was analyzed by Real-Time PCR, the result showed that 14-3-3 gene was most expressed in hepatopancreas and muscle, then in eyestalk, intestinal, heart and gill, the lest in stomach. After 24h’s stress in salinity, the expression of 14-3-3 gene increased great significantly (P<0.01) whatever the salinity reduced (5) or raise (15, 20, 25, 30). The more the salinity changed, the more the 14-3-3 gene expressed. These results will serve further studies on functions and regulation mechanism of 14-3-3 genes.

Abstract:To produce bioactive peptides from the by-products of fish processing, bigeye tuna (Thunnus obesus) head was hydrolyzed by alcalase, and the tuna head protein hydrolysate (THPH) was fractionated through ultrafiltration membranes with a range of molecular weight cutoffs (MWCO) of 8, 5, 3 and 1 ku, respectively, to yield the fraction THPH-1 with MW distribution<1 ku. The antioxidant activities of THPH-1were evaluated using reducing power, free radical scavenging and aged model mice induced by D-galactose. The physicochemical properties of THPH-1 were analysed also. THPH-1 showed evident radical scavenging activity in a dose-dependent manner with the IC₅₀ values for hydroxyl radical, superoxide radical and 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical being 1.38, 0.73 and 0.93 mg/mL respectively. The reducing power of THPH-1 was 0.763 at 12.5 mg /mL. The activities of superoxide dismutase (SOD) of liver and the activities of glutathione peroxidase (GSH-PX) of liver and serum in aged model mice were significantly increased (P<0.05) compared with that of the normal control group mice after 42 days administrated orally at a dose of 30 mg/kg THPH-1, and the content of malondialdehyde (MDA) of serum in aged model mice was significantly decreased (P<0.01).The physicochemical properties showed that THPH-1contained 96.40% of protein(on dry basis), 0.11% of fat and 4.86% of ash. The total hydrophobic amino acids content of THPH-1was 35.8% of the total amino acids content. The molecular weight distribution of the higher activity fractions of THPH-1were located at 1 802-2 519 u and 422-922 u.These results indicate that THPH-1from tuna head hydrolysate would be very fairly helpful in the preparation of antioxidative peptides and a beneficial ingredient for functional food.