Histopathological Observation of Lymphocystis Disease and Lymphocystis Disease Virus （LCDV） Detection in Cultured Diseased Sebastes schlegeli

Lymphocystis nodules occurring in the cultured sting fish Sebastes schlegeli were observed under light and electron microscope. Lymphocystis disease virus (LCDV) in the tissues of diseased fish was detected with indirect immunofluorescence test (IFAT). Results showed that lymphocystis cells had overly irregular nuclei, basophilic intracytoplasmic inclusion bodies with virions budding from the surface, and hyaline capsules outside the cell membrane. Numerous virus particles about 200 nm in diameter scat- tered in the cytoplasm, electron-dense particles 70-80 nm in diameter filled in perinuclear cisterna, and membrane-enveloped parti- cles with electron-dense core of 70-80 nm appeared around cellular nucleus. IFAT using monoclonal antibody against LCDV from Paralichthys olivaceus revealed that specific green fluorescence was present in the cytoplasm of lymphocystis cells, epithelium of stomach, gill lamellae, and muscular fibers under epidermis of S. schlegeli, just as that in the cytoplasm of lymphocystis cells of P. olivaceus, suggesting the presence of LCDV in these tissues.     

A NOTE ON CIRCULATION OF THE EAST CHINA SEA

The East China Sea lies over a wide continental shelf off the coast of China. Just like its neighbouring areas Bohai Sea and Huanghai Sea, the major part of the East China Sea is of a shallow water area, its mean depth being about 370 m. On the average, the annual discharge of fresh water into Bohai Sea, Huanghai Sea and the East China Sea is" about 1.5×10~(12)m~3, and among these, about 80％ is discharged into the East China Sea, the Changjiang River alone accounts for about 9×10~(11)m~3/year. Mixed with this large amount of fresh water the coastal water is of low salinity in this area. This Coastal Water System flows, generally, from north to south. Off the outer margin of the continental shelf, a powerful flow characterized by high temperature and high salinity and known as the Kuro-     

ALFISOLS AND CLOSELY RELATED SOILS IN CHINA

Alfisols are important soils in China. They occupy about 1.25 million km3, or about 13% of the land area. In the current Chinese system of soil classification, burozem, yellow-brown earths, Baijiang (Planosol) soils and parts of drab soils. They are mostly forested soils with an estimated 5-13 t / ha · yr of organic matter returned to the soils from temperate mixed conifer and broad-leaved forest. In terms of elemental bio-cycling, Ca is prominent.In a comparison of 30 profiles the average ratio of clay (B/ A) was 1.47 for Cryoboralfs and Eutroboralfs; 1.88 for Hapludaifs and 2.53 for Paleudalfs. From Eutroboralfs to Paleudalfs the average gain (or loss) in clay during soil development is about a factor of seven.The moisture regimes vary considerably between Hapludaifs, Cryoboralfs, and associated Cryaquepts, but the amount of water is always enough to cause significant leaching. In the weathering, and pedogenesis processes TiO2, MgO and Fe2O3 are accumulated, respectively, in both A and BA horizons; b     

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.     

Inhibition of photosynthesis in the microalga Chaetoceros curvisetus （Bacillariophyta） by macroalga Gracilaria lemaneiformis （Rhodophyta）

We investigated the effects of dried macroalga Gracilaria lemaneiform （Rhodophyta） on photosynthesis of the bloom-forming microalga Chaetoceros curvisetus. C. curvisetus was cultured with different amounts of dried G. lemaneiformis under controlled laboratory conditions. We measured the photosynthetic oxygen evolution rate and established the chlorophyll a fluorescence transient （OJIP） curve coupled with its specific parameters. We observed concentration-dependent and time-dependent relationships between dried G. lemaneiformis and inhibition of photosynthesis in C. curvisetus. Co-culture with dried G. lemaneiformis also resulted in a decrease in the light-saturated maximum photosynthetic oxygen evolution rate （P~ax） in C. curvisetus, and a decrease in the OJIP curve along with its specific parameters; the maximum photochemical efficiency of PSII （FJFm）, the amount of active PSII reaction centers per excited cross section at t=0 and t=--tFM （RC/CS0 and RC/CSm, respectively）, the absorption flux per excited cross section at t=0 （ABS/ CS0）, and the efficiency with which a trapped exciton moves an electron into the electron transport chain （~u0）. The dark respiration rate （Rd） increased in C. curvisetus co-cultured with dried G. lemaneiformis. The JIP-test and the oxygen evolution results indicated that dried G. lemaneiJbrmis decreased the number of active reaction centers, blocked the electron transport chain, and damaged the oxygen-evolving complex of C. curvisetus. This result indicated that dried fragments of G. lemaneiformis could effectively inhibit photosynthesis of C. curvisetus, and thus, could serve as a functional product to control and mitigate C. curvisetus blooms.     

Energy flow in Branchiura sowerbyi （Oligochaeta： tubificidae） in a shallow macrophyte-dominated lake, Biandantang Lake

The energy flow of Branchiura sowerbyi was studied for the first time in China in a shallow macrophytic lake, Biandantang Lake, Hubei Province. The energy flow was calculated from the measurement of flesh production (12.5241kJ/m^2a), egestion (517.7302kJ/m^2a), metabolism (38.3273 kJ/m^2a), and excretion (4.3798kJ/m^2a). The net growth efficiency of the species is about 22.7%, which accords well with the generally reported value for oligochaetes. In addition, the relationship between starvation respiration (R, mgO2/ind‘d), wet weight (Ww, mg) and temperature (T, ℃) were also measured, with the regression function being R=0.008 Ww^0.736 e^0.050T.     

Biomass and carbon storage of Gracilariopsis lemaneiformis （Rhodophyta） in Zhanshan Bay,Qingdao, China

Marine macroalgae can absorb carbon and play an important role in carbon sequestration. As an important economic macroalga, Gracilariopsis lemaneiformis has the potential to significantly affect carbon absorption and storage in wave-sheltered intertidal reef systems. However, detailed knowledge on seasonal biomass changes and carbon storage of G. lemaneiformis is lacking, especially in many small and scattered ecosystems. Considering the influence of human activities on wild distribution of G. lemaneiformis, the understanding of seasonal dynamics of an economically important species in nature is necessary. In this study, we first investigated seasonal variations in biomass, coverage area, and carbon storage during low tide from August 2011 to July 2012 in Zhanshan Bay, Qingdao, China. Furthermore, we estimated the carbon storage potential of wild G. lemaneiformis using light use efficiency(LUE). The results show that the standing biomass and coverage area changed significantly with season. However, seasonal variations in carbon content and water content were not obvious, with an average content of 35.1% and 83.64%, respectively. Moreover, carbon storage in individual months varied between 0.67 and 47.03 g C/m 2, and the value of carbon storage was the highest in August and June and the lowest in February. In Zhanshan Bay, LUE of G. lemaneiformis was only 0.23%. If it is increased to the theoretical maximum(5%–6%), the carbon storage will have an increase of at least 21 times compared with the current, which suggested that carbon storage of wild G. lemaneiformis had a high enhancement potential. The study will help to assess a potential role of G. lemaneiformis in reducing atmospheric CO2.     

Characterization,tissue distribution,and expression of neuropeptide Y in olive flounder Paralichthys olivaceus

Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture.     

UP-REGULATION OF HEPATIC RECEPTOR FOR GROWTH HORMONE IN THE FLOUNDER （PARALICHTHYS OLIVACEUS） AFTER ORAL ADMINISTRATION WITH EXOGENOUS GH

The iodination efficiency of salmon GH (sGH) was 38.82%,using a modification of the chloramine-T method. The specific activity of the ^125I-sGH was about 40 μCi/μg protein. The results of binding assay showed a single class of high affinity and low-capacity binding site in flounder liver. Long-term administration with exogenous GH can induce the up-regulation of hepatic GH receptor in total binding capacity though there was no significant difference of association constant among any groups. Con-sidering that there was no significant difference in capacity of free binding sites of livers from control and experimental fish, this result also indicated that the liver from experimental fish, compared to that from control fish, had more occupied binding sites.     

淋巴囊肿病毒中国分离株基因组序列和结构分析

LCDV-cn是分离自中国养殖牙鲆的淋巴囊肿病病原,给中国牙鲆养殖业带来了严重危害。通过PCR、克隆和测序,获得了LCDV-cn部分基因的序列,证明LCDV-cn与LCDV-C的基因组序列是一致的。比较基因组学分析发现,LCDV-cn基因组中有137个ORF是其独有的、与LCDV-1及其它虹彩病毒无同源区域。对LCDV-cn基因组进行生物信息学分析发现,在LCDV-cn独有的ORF中,有的编码与宿主同源的蛋白,有的编码与DNA复制相关的蛋白,某些ORF编码的蛋白含有跨膜区等。LCDV-cn的独有基因或许对其毒力和宿主范围有关。     

Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>) tissues

Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass (Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of iymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector.Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.     

Localization and Dynamic Expression of a 27.8kDa Receptor Protein for Lymphocystis Disease Virus Infection in Sea Bass(Lateolabrax japonicus) Tissues

Lymphocystis disease virus(LCDV) infects target cells by attaching to a 27.8 k Da receptor(27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies(MAbs) have been developed. However, the 27.8R existence in tissues of sea bass(Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder(Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also ana...     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.     

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.     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)