Effect of artificial regulations ofArtemia n−3 HUFA content on growth and survival of black seabream (Sparus macrocephalus) larvae

The requirement for dietary n−3 HUFA (highly unsaturated fatty acid) for growth and survival of black seabream (Sparus macrocephalus) larvae was tested usingArtemia with various levels of n−3 HUFA. Four treatments withArtemia differing in their n−3 HUFA were prepared by feeding them various oil emulsions. The results indicated that dietary n−3 HUFA significantly influences fish n−3 HUFA levels and are necessary for good growth and survival of black seabream larvae. The proper value of n−3 HUFA level and DHA, EPA level inArtemia should be 4.273% and 0.873% (wet weight) for good growth and survival in black seabream larvae respectively. Contribution No. 3044 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39470562 supported by NSFC.     

A successful microbound diet for the larval culture of Chinese shrimp Fenneropenaeus chinensis

A 13-day feeding trial was carried out to evaluate the effectiveness of a microbound diet for rearing the larvae of Chinese shrimp Fenneropenaeus chinensis in comparison with the live foods that consist of Isochrysis galbana, Chlorella vulgaris, Tetraselmis chuii, rotifer (Brachionus plicatilis) and Artemia sp. Larvae of 0 to 13d post-hatch (dph) were reared in a temperature-controlled semi-open culture system and stocked at a density of 100 larvae L⁻¹ in tanks, each containing 50 L sterilized seawater with salinity 30–32. Larvae were manually fed either the live foods or the microbound diet 6 times per day. At 13dph, the growth of the larvae fed on the microbound diet was approximately 84% of that fed on the live foods. The survival rate of the larvae fed on the microbound diet was 44.29% at 13 dph, which was not significantly different from that of larvae fed on live foods (63.55% ). The body length and development index (DI) of the larvae fed on the microbound diet were always lower than those of larvae fed on live foods. However, the differences reached significant levels only at 11 and 13dph (P<0.05). The mean dry weight loss of the microbound diet was 9.2% after 90min immersion in seawater, indicating that this diet has a good water stability. The microbound diet contains 5223% crude protein and 10.27% lipid and is easy to prepare. These characteristics of the diet suggest good potentials for its successful use in the larviculture of other penaeid and fish species.     

The effects of temperature and substrate on ontogenetic behavior of bastard halibut, Paralichthys olivaceus （T.et S.）

The effects of temperature and substrate on ontogenetic patterns and settlement behavior of bastard halibut, Paralichthys olivaceus （T. et S.） were studied in a series of laboratory experiments. Analysis on stage-based data revealed that except for settlement, and compared to the substrate, the ambient temperature dominantly controlled the hatch success and subsequent development of the species. The oosperm optimum survival rate of 60% during hatching occurred at 20℃, and survival rate of 29%, at 18℃ for larval rearing. The survival rate of larvae reared under variable temperature of 3.5-4.5℃ was higher than that of those reared under stable temperature. The maximum survival rate of 29%, corresponded to variation at temperature of 4℃. With increase in age newly settled juveniles preferred to lie on the fine substrate and gradually moved on the coarse substrate. GLM （general linear model） analysis showed that the combination of temperature and substrate had no significant impact on the survival of settling larvae, but definitely affected the duration of settlement.     

Effects of dietary soy isoflavones on feed intake, growth performance and digestibility in juvenile Japanese flounder (Paralichthys olivaceus)

An 8-week feeding trial was conducted to investigate the effects of dietary soy isoflavones on feeding intake,growth performance,and digestion of juvenile Japanese flounder(Paralichthys olivaceus).Four isonitrogenous(49% crude protein) and isoenergetic(20.1 MJ kg-1) diets were formulated to contain four graded levels of soy isoflavones,namely,0,1,4 and 8 g soy isoflavones in 1 kg of diet.Each diet was randomly fed to triplicate tanks of fish(Initial average weight:2.58 g ± 0.01 g),and each tank was stocked with 35 fish.No significant difference was observed among diets with levels of 0,1 and 4 g kg-1 soy isoflavones in feed intake,weight gain,feed efficiency ratio(FER),proximate composition of fish whole body and apparent digestibility coefficients(ADC) of nutrients and energy(P>0.05).However,high dietary soy isoflavones level(8 g kg-1) significantly depressed weight gain,FER,whole-body crude lipid content of fish and ADC of nutrients(P<0.05).These results indicate that high level of dietary soy isoflavones(above 4 g kg-1) significantly depresses growth responses and FER of Japanese flounder.However,as the content of soy isoflavones in soybean meal is around 1 to 3 g kg-1,the adverse effects might be neglected when soybean products are used as a fish feed ingredient.     

Histological study on the skin of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Histological development of Japanese flounder Paralichthys olivaceus larval skin and ultrastructural difference of skin between reared normal and malpigmented Japanese flounder were studied with light microscopy (LM) and transmission electron microscopy (TEM). The results show that the skin develops slowly before the metamorphosis, while at the onset of metamorphosis, the skin develops quickly and becomes complete in structure till about 50d after being hatched. Ultrastructural observation on the normal and malpigmented skins shows that the iridophore and melanophore are adjacent to each other. Profile and structure of the two kinds of pigmcnt cells are more complete in the skin of normal ocular side than in the skin of pigmented blind side. The ultrastructure of typical chloride cell was observed in the skin of Japanese flounder larvae for the first time.     

Analysis of IL-2-1ike factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%. This work was supported by National “973” Project G1999012003, G19999012006.     

Steroid sex hormone dynamics during estradiol-17β induced gonadal differentiation in Paralichthys olivaceus (Teleostei)

Steroid sex hormones, such as estradiol-17β (E2) and testosterone (T), are important regulators of sex change in fish. In this study, we examined the effects of E2 treatment on the dynamics of E2 and T during gonadal differentiation in the olive flounder Paralichthys olivaceus using histology and radioimmunoassay (RIA). Flounder larvae were divided into five groups (G0-G4), and fed with 0 (control), 0.2, 2, 20 and 100 mg E2/kg feed from 35 to 110 day post hatching (dph). Fish growth in the G1 and G2 groups was not significantly different from that of the control group (P>0.05), while fish in the G3 and G4 groups were less active and showed growth depression and high mortality. The gonads of fish in the G3 and G4 groups were smaller and surrounded by hyperplastic connective tissue. The frequency of females in the G0-G4 groups was 54.5%, 75.0%, 100%, 100% and 93.3%, respectively. The RIA analyses of E2 and T showed that T levels decreased during gonadal differentiation, and increased slightly at the onset of ovarian differentiation, while E2 levels increased gradually and peaked at the onset of ovarian differentiation in the control group. In the E2-treated groups, T levels decreased before the onset of ovarian differentiation. E2 levels were high on the 48 dph, but declined to a lower level on the 54 dph, and then increased gradually during gonadal differentiation. And a sharp increase of E2 levels were observed in all E2-treated groups at the onset of ovarian differentiation. The data suggest that T and E2 play important roles during gonadal differentiation, and an E2 dose of 2 mg/kg feed could induce sex reversal in P. olivaceus.     

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus： karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)     

A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infec Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese floun were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Prot spots demonstrating changes greater than two-fold in the expression level were digested and furt identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immuni relevant proteins were thus identified as transferrin and the complement component C3 of Japan flounder. These findings suggest that the two proteins may play important roles in the self-healing lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing LCDV-infected Japanese flounder by improving their innate immunity.     

Development of retinal structure and visual acuity in Japanese flounder (Paralichthys olivaceus)

The retinal structure and visual acuity in Japanese flounderParalichthys olivaceus at different stages of development were examined by light microscopy. The resolving power of the retina, the visual axis and the best visual field were estimated based on the distribution of cone cells in the retina. The visual system of the larvae appears poorly developed at hatching. The larvae with total length (TL) of less than 10 mm, have single cones only and the eyes were well pigmented. At 10–11 mm TL, most single cones fused to form double cones, with the single and double cones forming a mosaic pattern. From larvae to early juvenile the retina stretches, the cones increase in diameter and rods increase in number. Based on the highest density of the cones in the ventro-temporal region, the visual axis was orientated upforward. The resolving power of the retina in 40–530 mm TL Japanese flounder was found to range from 25.1 to 11.5 min. The results indicated continual improvements in the visual system of the growing fish towards higher resolving power, visual acuity and sensitivity. Project 39970578 supported by the NSFC, and also supported by the Ministry of Education Foundation for University Key Teachers.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder <Emphasis Type="Italic">Platichthys stellatus</Emphasis>

Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.     

Ontogeny of the lymphoid organs of Japanese flounder, Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40 d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells were observed within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.     

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405), we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.     

Effect of n?3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream ( Sparus macrocephalus )

Requirement for dietary n−3 HUFA (n−3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers andArtemia at various levels of n−3 HUFA. Five treatments with rotifers andArtemia differing in n−3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n−3 HUFA significantly influence fish n−3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n−3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n−3 HUFA fatty acid ethyl esters from ethyl-esterified oil. Project No. 39770588 supported by the NSFC.     

饲料中添加L-肉碱对牙鲆幼鱼生长、生化组成及血液指标的影响

研究0、300、600、900、1 200、1 500 mg/kg 6个L-肉碱添加水平对牙鲆(Paralichthys olivaceus)幼鱼生长、生化组成和血液指标的影响。结果表明:添加量为1 200 mg/kg的处理组牙鲆幼鱼的增重率和特定生长率显著提高,饲料系数显著降低(P<0.05);随着饲料中L-肉碱含量的增加,肝指数呈现下降趋势,且在添加量为900~1 500 mg/kg时,各组较对照组差异显著(P<0.05);饲料中添加L-肉碱显著降低各实验组脏体比(P<0.05),而对各处理组间存活率和肥满度均无影响(P<0.05);肌肉和肝脏中的脂肪含量与L-肉碱添加量呈负相关,当添加量为1 200~1 500 mg/kg时,较对照组差异显著(P<0.05);肌肉中的蛋白含量与L-肉碱添加量呈正相关,但各组差异不显著(P<0.05);饲料中添加L-肉碱降低血清中的谷草转氨酶、胆固醇、甘油三酯和尿素氮含量,且添加量1 200 mg/kg组各指标含量均显著低于对照组(P<0.05);各处理组的血糖含量较对照组均有所增加,其中1 200 mg/kg组血糖含量较对照组提高41.6%(P<0.05);血清中总蛋白和低密度脂蛋白均随着L-肉碱添加量的增加而呈下降趋势,高密度脂蛋白却得以提升,但差异均不显著(P<0.05)。分析认为,在本实验条件下,饲料中添加L-肉碱可促进牙鲆幼鱼生长,且降脂效果显著。建议L-肉碱最适添加量为1 200 mg/kg。     

Properties of proteolytic toxin of Vibrio anguilolarum from diseased flounder

Extracellular products (ECP) produced byVibrio anguillarum strain M3 originally isolated from diseased flounder (Paralichthys olivaceus) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M2 showed highest growth and protease activity at 25°C. The protease present in ECP showed maximal activity at pH 8 and 55°C; was completely inactivated by application of 80°C heat for 30 min; was completely inhibited by EDTA and HgCl₂, and was partially inhibited by PMSF, SDS, MnCl₂ and iodoacetic acid; but not inhibited by CaCl₂ and MgCl₂. The ECP was toxic to flounder fish at LD₅₀ values of 3.1 μg protein/g body weight. The addition of HgCl₂ and application of heat at 50°C decreased the lethal toxicity of ECP. When heated at 100°C, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerious lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacteriumV. anguillarum M3. Contribution No. 4213 from the Institute of Oceanology, Chinese Academy of Sciences. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

FURTHER OBSERVATION OF THE SPERMATOZOA OF LEFT-EYE FLOUNDER PARALICHTHYS OLIVACEUS BY ELECTRONIC MICROSCOPY

The spermatozoon ultrastrucmre of left-eye flounder, Paralichthys olivaceus, was ob-served by electronic microscopy, and shown to consist of head, midpiece and tail. Some nuclear vacuoles were observed inside and along the outer part of the nucleus and double-membrane structures were ob-served between the nuclear membrane and plasma membrane. The sperm sleeve consisted of four indepen-dent parts and had more than four mitochondria. Along the sperm tail there were wing-like lateral tins.     

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.