Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Establishment,characterization of a new cell line from heart of half smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

A new cell line was established from the heart of a cultured marine fish, half smooth tongue sole (Cynoglossus semilaevis), designated as CSH (Cynoglossus semilaevis heart cell line). The CSH cells grow over 400 days in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 2 ng/ml basic fibroblast growth factor (bFGF). The suitable temperature for the cell growth was 24–30°C with the optimum growth at 24°C and a reduced growth at 12 and 30°C. FBS and bFGF concentration were the two important components for CSH cells proliferation. Twenty percent FBS in the medium was found to be the optimum concentration and bFGF promoted the growth of CSH cells. The double time of the cells at 24°C was determined to 73.39 h. Chromosome analysis revealed that 44% of the cells maintained a normal diploid chromosome number (2n = 42) in the CSH cells at Passage 58. The fluorescent signals were observed in CSH after the cells were transfected with green fluorescent protein (GFP) reporter plasmids. CSH cells showed the cytopathic effect (CPE) after infection with lymphosystis disease virus (LCDV). Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy, and a segment of MCP gene for major capsid protein of LCDV was found by PCR amplification DNA of virus-infected cells.     

New methods for the primary culture of gill epithelia from freshwater rainbow trout

Gill epithelia from freshwater rainbow trout can be grown in primary culture in Leibovitz's L-15 medium, by seeding freshly isolated gill cells on two successive days, from two different fish, directly onto permeable filter supports (DSI technique). This preparation allows the measurement of transepithelial resistance (TER) and exposure of the apical surface to freshwater, as in vivo. New culture methods were developed and evaluated, using TER as an indicator of epithelial integrity, in an effort to improve the utility of the preparation for proteomic and toxicological research. TER was not related to cell density or protein content in DSI epithelia. To eliminate bovine proteins, the 5% foetal bovine serum (FBS) normally required for epithelial development was replaced with trout plasma. While previously frozen trout plasma proved toxic, freshly collected heparinized plasma, provided by chronically cannulated adult trout, was not. The use of 5% fresh trout plasma supported a TER development curve identical to that with 5% FBS, a useful advance for proteomic research because foreign (bovine) proteins are eliminated. However, 10% plasma reduced TER development, and 100% plasma abolished it. The inhibitory effect on TER of high plasma levels was seen only early in epithelial development, and was exerted from the apical side, likely an effect on tight junction formation. Mature plasma-supplemented preparations mounted a TER rise in response to apical freshwater exposure comparable to that of FBS-supplemented epithelia. Yolk-sac fry extract was inhibitory to TER development, even in the presence of 5% FBS. Transfer of mature epithelia from 18 °C to 4 °C maintained stable TER and extended the useable lifespan by at least ten days, thereby facilitating storage of preparations for toxicity testing. A new method of growing epithelia, involving only a single seeding of cells from a single fish, directly onto filter inserts (SDSI technique), provided mature epithelia with much lower TER, a smaller TER response to apical freshwater, and lower cell density and protein content than DSI epithelia. These SDSI epithelia offer the advantage of multiple preparations grown directly from unique individuals for in vitro toxicity testing. This revised version was published online in July 2006 with corrections to the Cover Date.     

塘鳢的养殖试验

塘鳢是淡水小型经济鱼类，江浙沪一带将其视为名菜，为发展特种小水产品，作者于１９９２～１９９５年进行了塘鳢养殖试验工作，取得了人工繁殖，苗种培育及成鱼饲养的成套经验，人工繁殖和苗种培育在２０ｍ＾２的网箱中进行，鱼孵化率和苗种（夏花）成活率分别达到５９．７１％和７１．０３％，成鱼饲养采取池塘混养方式，在２６７ｍ＾２的池塘中经１７０天养成尾重２０ｇ以上的商品鱼１８．２５ｋｇ，成鱼饲养成活率为６４．５％     

Establishment and characterization of a kidney cell line from kelp grouper <Emphasis Type="Italic">Epinephelus moara</Emphasis>

A novel cell line, Epinephelus moara kidney cell line (EMK), was established from kidneys of kelp grouper E. moara. Cells were cultured at 24 °C in Leibovitz’s L-15 medium (L15) supplemented with antibiotics, basic fibroblast growth factor (bFGF), foetal bovine serum (FBS) and 2-mercaptoethanol (2-ME). EMK cells, fibroblastic in morphology, proliferated to 100% confluency in 3–4 days and were subcultured for over 50 passages. The cells could grow from 18 to 30 °C, with optimal growth at 24 °C. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 42. Green fluorescent signals could be observed in EMK cells when the cells were transfected with pEGFP-N3 plasmid. Moreover, a significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or nervous necrosis virus (NNV), and viral replication was confirmed by quantitative real-time PCR (qPCR). These results suggested the potential of the EMK cell line for studies of transgene and pathogenesis of SGIV and NNV.     

Establishment and characterization of a fin tissue cell line derived from silver pomfret,Pampus argenteus

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT‐PCR and viral titre assays. In contrast, PaF cells were resistant to red‐spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune‐related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen–silver pomfret interaction.     

Thermal Optima for the Culture of Juvenile Summer Flounder,Paralichthys dentatus

Abstract

Summer flounder, Paralichthys dentatus, aquaculture has shown promise over the recent past and a considerable body of knowledge is amassing for this species of flatfish. Even with the amassing data, basic information on environmental parameter ranges to maintain in indoor culture systems is still lacking. Therefore, the current study was undertaken to elucidate a temperature range that may be best suited for juvenile summer flounder production in indoor recirculating aquaculture. A 10-week study was designed to address 19, 24 and 29°C as potential temperatures for the culture of summer flounder. Fish averaging 9.5 g initial weight were stocked into triplicate 20-L aquaria per treatment after conditioning to their respective temperatures for 1 week. Fish were fed to apparent satiation twice daily a 50% crude protein fingerling diet and weighed every 2 weeks to assess growth rates.

Juvenile summer flounder grew better at 24 and 29°C (167 and 197% increase initial weight, respectively) than at 19°C (97% increase in initial weight) with increased individual fish variation within a treatment as temperature increased above 19°C. Feed efficiency was greatest at 24 and 29°C (0.65 and 0.57, respectively), but survival decreased at temperatures above 19°C (93, 60 and 57% for 19, 24 and 29°C, respectively). There was no effect of temperature on the hepatosomatic index or interior muscle ratio, but the finray muscle ratio was slightly elevated when flounder were cultured at 29°C. The lipid content of the finray muscle and liver also increased at 29°C. Therefore, 24°C appears to be the best culture temperature for summer flounder with respect to growth rates and efficiency, but survival and homogeneity of flounder may be lowered at temperatures above 19°C.     

池塘培育大眼狮鲈鱼苗、鱼种的试验

哈尔滨市水产研究所于1993年5月24日从加拿大FortQu′Appelle鱼苗孵化场移运大眼狮鲈鱼发现鱼卵，经孵化后孵出鱼苗14万尾，在池塘中培育。鱼苗阶段培育36天，体长53．9mm，成活率75％；鱼种阶段经95－100天培育，套养的鱼种体长20－22．5cm，体重83－107．4g，单养的鱼种体长17．5cm，体重48．4g。     

Development and characterization of a new marine fish cell line from turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n = 44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.     

Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

Effect of dietary protein, lipid and carbohydrate ratio on growth, feed efficiency and body composition of pikeperch Sander lucioperca fingerlings

Pikeperch Sander lucioperca fingerlings were fed nine practical diets containing three levels of protein (P=34%, 43% and 50%), lipid (L=10%, 16% and 22%) and carbohydrate (C=10%, 15% and 20%) for 10 weeks in a recirculating water system at 23°C. Dietary treatments were distributed by orthogonal design with dietary energy content ranging from 15.5 to 23.1 MJ kg^?1 diet. Significant differences (P<0.05) in weight gain (%) and feed efficiency (FE) were observed after feeding trial. Relatively low growth and FE were found in fish fed diets containing 34% dietary protein level compared with that of fish fed diets with 43–50% protein levels, suggesting that 34% dietary protein probably is below the protein requirements of pikeperch fingerlings. Fish fed diets containing P43L10C15, P43L22C20 and P50L16C20 had significantly (P<0.05) higher weight gain and FE than fish fed the diets containing other dietary P/L/C ratios. There was no significant difference in weight gain and FE between fish fed diets of P43L10C15, P43L22C20 and P50L16C20. These results may indicate that pikeperch require at least 43% of dietary protein for adequate growth and FE, and considering the fish growth and feed ingredient cost P43L10C15 diet is more cost‐effective formulation for pikeperch fingerling. However, protein efficiency was not significantly affected by dietary P/L/C ratio.     

Dietary Arginine Requirement of Fingerling Indian Major Carp,Catla catla (Hamilton)

Dietary arginine requirement of fingerling Catla catla (3.55 ± 0.05 cm; 0.61 ± 0.02 g) was determined by feeding casein–gelatin‐based isonitrogenous (33% crude protein) and isocaloric (3.40 kcal/g digestible energy) amino acid test diets containing six graded levels of l ‐arginine (1, 1.25, 1.5, 1.75, 2, and 2.25% dry diet) for 12 wk. Maximum absolute weight gain (6.93 g/fish), protein efficiency ratio (2.13), protein deposition (0.36), arginine retention efficiency (78%), and best feed conversion ratio (1.42) were recorded in fish fed 1.75% arginine of the dry diet. Maximum carcass protein (15.57%) and RNA/DNA ratio (4.79) were also recorded for the group fed 1.75% arginine of the dry diet. Quadratic regression analysis at 95% maximum or minimum response of above growth parameters yielded optimum arginine requirement of fingerling C. catla at 1.67% of the dry diet. On the basis of the above analysis of the growth parameters, it is recommended that the inclusion of dietary arginine at 1.67% of the dry diet is optimum for formulating arginine‐balanced, cost‐effective quality feeds for the mass culture of fingerling C. catla .     

Substitution of fish meal with raw or treated cowpea (Vigna unguiculata L Walp,IT86‐D719) meal in diets for Nile tilapia (Oreochromis niloticus L.) fry

Two feeding experiments were carried out to evaluate the effect of partial substitution of fish meal (FM) with raw or heat‐treated cowpea Vigna unguiculata L. Walp var. IT86‐D719 seeds on growth performance, digestibility and pancreas tissue in Nile tilapia (Oreochromis niloticus L.) fry. Experiment 1 involved six isonitrogenous and isocaloric diets: four containing different concentrations of raw, whole cowpea meal (protein basis), a positive control with FM as sole protein source and a negative with cowpea meal as sole protein source. Substitution at up to 200 g kg^?1 had no significant effect on production parameters, although growth was negatively affected in the negative control because of seed antinutritional factor content. Experiment 2 evaluated the effect of heat treatment (oven drying at 48 °C; hot air drying at 70 °C; or autoclaving at 119 °C) and/or seed dehulling using diets containing 200 g kg^?1 cowpea meal presoaked in water. Only autoclaving eliminated trypsin inhibitor and lectin contents, independent of dehulling. Histological analysis indicated no histological changes in pancreas tissue. Raw or treated cowpea meal can replace FM in tilapia fingerling diets at up to 200 g kg^?1 protein content without significantly affecting productive parameters or pancreas tissue.     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Establishment of a continuous cell line from female half‐smooth tongue sole (Cynoglossus semilaevis)

A new cell line (TSHC) derived from heart tissues was established from female half‐smooth tongue sole (Cynoglossus semilaevis), an economically important marine fish species in China. The cell line had been subcultured for more than 30 times over a period of 200 days. The cell line was optimally maintained at 24°C in minimum essential medium (MEM) medium containing foetal bovine serum (FBS), 2‐mercaptoethanol (2‐Me), sodium pyruvate, basic fibroblast growth factor (bFGF) and antibiotics. The TSHC cells were mostly composed of fibroblast‐like cells. Chromosome analysis revealed that the TSHC cell line had a normal diploid karyotype with 2n = 42, containing the heterogametic W chromosome. The TSHC cell line was susceptible to infection by flounder Lymphocystis disease virus (LCDV). Although an atypical cytopathic effect and only few of virus particles in the cytoplasm was observed, it provides a research material on the cell–pathogen interaction research about the viral infection of non‐host species.     

Establishment of a new fish cell line from the brain of humpback grouper (Cromileptes altivelis) and its application in toxicology and bacterial susceptibility

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26 °C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.

     

Impact of Aeration on the Growth Performance of Silver Barb,Puntius gonionotus,during Fingerling Rearing

Impact of aeration on growth of silver barb, Puntius gonionotus during fingerling rearing was studied through a 100‐d rearing experiment conducted in 18 concrete tanks of 50 m² (10 × 5 × 1.2 m) size. Fry (0.74 ± 0.27 g, 35 ± 6 mm) were stocked in the experimental tanks at three stocking densities (25, 50, and 75 fry/m²) and were evaluated with and without provision of 6 h (2400–0600 h) of night time aeration. Aeration resulted in higher pH and dissolved oxygen regime and increased fingerling length and weight. The results suggest a rearing density of 75/m² to be ideal for rearing fry to fingerling of this species when aeration is provided, whereas, under non‐aerated condition, rearing the fry to fingerling stage at 50/m² was found advantageous over those at 25 and 75/m².