Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a vitally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx wash also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Effects of the "all-fish" growth hormone transgene expression on non-specific immune functions of common carp, Cyprinus carpio L.

This study investigated non-specific immune functions of the F-2 generation of "all-fish" growth hormone transgenic carp, Cyprinus carpio L. Lysozyme activity was 145.0 (+/- 30.7) U ml(-1) in the transgenic fish serum and 105.0 (+/- 38.7) U ml(-1) in age-matched non-transgenic control fish serum, a significant difference (P < 0.01). The serum bactericidal activity in the transgenics was significantly higher than that in the controls (P < 0.05), with the percentage serum killing of 59.5% (6.83%) and 50.8% (8.67%), respectively. Values for leukocrit and phagocytic percent of macrophages in head kidney were higher in transgenics than controls (P < 0.05). However, the phagocytic indices in the transgenics and the controls were not different. In addition, the mean body weight of the transgenics was 63.4 (6.65) g, much higher than that of the controls [39.2 (+/- 3.30) g, P < 0.01]. The absolute weight of spleen of the transgenics [0.13 (+/- 0.03) g] was higher than that of the controls [0.08 (+/- 0.02) g, P < 0.01]. However, there was no difference in the relative weight of spleen between the transgenics and the controls, with the spleen mass index being 0.21% (+/- 0.02%) and 0.20% (+/- 0.03%), respectively. This study suggests that the "all-fish" growth hormone transgene expression could stimulate not only the growth but also the non-specific immune functions of carp. (c) 2006 Published by Elsevier B.V.

Acrossocheilus spinifer, a new species of barred cyprinid fish from south China (Pisces : Teleostei)

Acrossocheilus spinifer sp. nov. is described from the river basins in Fujian Province and the Han Jiang basin in Guangdong Province, south China. It is one of the barred Acrossocheihis species sharing five or six vertical bars on the flanks, with each bar being two scales in width. Acrossocheilus spinifer sp. nov. is morphologically most similar to Acrossocheilus wenchowensis. but distinguished from it by: a colour pattern of all vertical bars extending ventrally to the second scale row below the lateral line, and having the second bar placed posterior to the base of the last simple dorsal-fin ray, a blunt snout; and a stout, last simple dorsal-fin ray. Acrossocheilus spinifer sp. nov. resembles Acrossocheilus kreyenbergii and Acrossocheilus stenotaeniatus with which it shares a stout, last simple dorsal-fin ray with a serrated posterior edge, but is separated from both species by having a narrow median interruption in the lower lip, a lower jaw fully covered by the lower lip when viewed ventrally and the second vertical bar placed posterior to the base of the last simple dorsal-fin ray. The absence of a longitudinal stripe extending along the lateral line on the flanks in males readily distinguishes A. spinifer sp. nov. from Four other species, namely Acrossocheilus fasciatus, Acrossocheilus paradoxus, Acrossocheilus parallens, and Acrossocheilus jishouensis. (c) 2006 The Authors Journal compilation (c) 2006 The Fisheries Society of the British Isles.

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes : Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post-hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood-thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3' untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5' flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. (c) 2005 Elsevier B.V All rights reserved.

Distribution, feeding and body condition of four small fish species in the near-shore and central areas of Liangzi Lake, China

Small fish abundance is usually high in heavily vegetated habitats in Yangtze lakes, China. Visual and swimming barriers created by dense macrophytes beds could reduce feeding efficiency and growth of small fishes. We tested the hypothesis that small fishes in habitats with dense macrophytes would show decreased feeding efficiency and reduced growth rates by comparing feeding efficiency (measured as the relative weight of fore-gut contents), total length, and condition factor of four small young-of-the-year fishes collected in the near-shore (heavily vegetated) and central (less vegetated) areas of Liangzi Lake. Feeding efficiency, total length, or condition factor were each significantly reduced in the near-shore area compared with the central area for Ctenogobius giurinus, Pseudorasbora parva and Carassius auratus auratus. This supports our hypothesis that vegetation abundance may mediate feeding efficiency and growth of small fishes. Although Hypseleotris swinhonis did not show significant decreases in feeding efficiency or growth in the near-shore area, there was not any reversed tendency, i.e. increased feeding rate or growth in the near-shore area compared to the central area.

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in delta(13)C and delta(15)N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in delta(13)C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in delta(13)C and delta(15)N of seston between surface and overlying waters. Spatially, the relatively high delta(13)C and delta(15)N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9-4.1 (3.5 +/- 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.

Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs. (C) 2004 Elsevier Ltd. All rights reserved.

Effect of replacement of fish meal by meat and bone meal and poultry by-product meal in diets on the growth and immune response of Macrobrachium nipponense

The potential use of poultry by-product meal (PBM) and meat and bone meal (MBM) as alternative dietary protein sources for juvenile Macrobrachium nipponense was studied by a 70-day growth trial. Triplicate groups of M. nipponense (initial body weight: 0.37 g) were fed at 20.7-22.4 degreesC on each of the five isoenergetic and isonitrogenous diets (protein content about 38%) with different replacement of fish meal by MBM or PBM. The control diet used white fish meal as the sole protein source, the other four diets were prepared with 15% or 50% fish meal protein substituted by either MBM (MBM15, MBM50) or PBM (PBM15, PBM50). The results showed that replacement of fish meal by MBM in diets did not affect growth performance of M. nipponense (P > 0.05), while specific growth rate in PBM15 was significantly higher than that in other groups (P < 0.05). Survival rates of shrimp fed with MBM15 diet were significantly higher than that in other groups (P < 0.05). No significant differences in immunological parameters, including total haemocyte count (THC), phenoloxidase activity (PO) and respiratory burst (O-2(-)), were observed between the shrimps that were fed five experimental diets, and all determined immunological parameters in control groups were slightly higher than those in replacement groups. In conclusion, either MBM or PBM investigated could replace up to 50% fish meal protein in diets for M. nipponense. (C) 2003 Elsevier Ltd. All rights reserved.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp (Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp (Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.

Small fish communities in two regions of the Liangzi Lake, China, with or without submersed macrophytes

Small fish communities were compared between the vegetated and vegetation-free regions of the Liangzi Lake, a shallow lake along the middle reach of the Yangtze River, China. Fish were sampled using 10 x 10 m(2) block nets and poisoning. Three samples were taken from either the near shore area or lake centre of each region. A total of 19 fish species were collected; all species occurred in the vegetated region but only 12 occurred in the vegetation-free region. The dominant small fish were Carassius auratus auratus in the vegetated region and Ctenogobius giurinus in the vegetation-free region. Diversity, density and biomass of small fishes were significantly higher in the vegetated region than in the vegetation-free region in both near shore and lake centre areas. In the vegetated region, density and biomass of small fishes was significantly higher, while species diversity significantly lower in the near shore area than in the lake centre. In the vegetation-free region, density of small fishes was significantly higher in the near shore area than in the lake centre area, but species diversity or biomass was unaffected by location.

Effect of several feeding stimulants on diet preference by juvenile gibel carp (Carassius auratus gibelio), fed diets with or without partial replacement of fish meal by meat and bone meal

The objectives of this work were to study the effects of several feeding stimulants on gibel carp fed diets with or without replacement of fish meal by meat and bone meal (MBM). The feeding stimulants tested were betaine, glycine, L-lysine, L-methionine, L-phenylalanine, and a commercial squid extract. Three inclusion levels were tested for each stimulant (0.18, 0.5%, and 1% for betaine and 0.1, 0.25 and 0.5% for the other stimulants). Two basal diets (40% crude protein) were used. one with 26% fish meal (FM), and the other with 21% fish meal and 6% MBM, Betaine at 0.1% in the fish meal group and at 0.5% in the meat and bone meal group was used in all experiments for comparison among stimulants. In the experiment on each stimulant, six tanks of fish were equally divided into two groups, one fed the FM diet, and the other fed the MBM diet. After 7 days' adaptation to the basal diet, in which the fish were fed to satiation twice a day, the fish were fed for another 7 days an equal mixture of diets containing varying levels of stimulants. Each diet contained a unique rare earth oxide as inert marker (Y2O3, Yb2O3, La2O3, Sm2O3 or Nd2O3). During the last 3 days of the experiment, faeces from each tank were collected. Preference for each diet was estimated based on the relative concentration of each marker in the faeces. Gibel carp fed the FM diet had higher intake than those fed the MBM diet, but the difference was significant only in the experiments on betaine, glycine and L-methionine. None of the feeding stimulants tested showed feeding enhancing effects in FM diets. All feeding stimulants showed feeding enhancing effects in MBM diets. and the optimum inclusion level was 0.5% for betaine, 0.1% for glycine, 0.25% for L-lysine, 0.1% for L-methionine. 0.25% For L-phenylalanine. and 0.1% for squid extract. The squid extract had the strongest stimulating effect among all the stimulants tested. (C) 2001 Elsevier Science B.V. All rights reserved.