National Laboratory for Marine Science and Technology

Qingdao, China

National Laboratory for Marine Science and Technology

Qingdao, China
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Zhang Q.,Chinese Academy of Fishery Sciences | Zhang Q.,National Laboratory for Marine Science and Technology | Zhang Q.,Shanghai Ocean University | Liu S.,Chinese Academy of Fishery Sciences | And 12 more authors.
Journal of Invertebrate Pathology | Year: 2015

A disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in China and several countries of Southeast Asia, leading to serious losses in production. Litopenaeus vannamei (also known as Pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. A new nodavirus, termed covert mortality nodavirus (CMNV), has been shown to be the etiological agent. In this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and quantitative detection of CMNV. The optimal conditions for this newly developed RT-LAMP reaction were found to be 6mM MgCl2 and 1.6mM dNTPs, an incubation temperature of 65°C and a reaction time of 50min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3pg total RNA of CMNV-infected shrimp and 27 copies of the target plasmid. The diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription PCR (RT-PCR) assay was 96.4% and 94.4%, respectively. The reaction products were detected by visual inspection after staining with an in-tube DNA fluorescent dye, a measure taken to eliminate the risk of contamination. The quantitative RT-LAMP assay for CMNV showed high correlation coefficient (r 2 =0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5×102 to 6.7×106 copies per mg of cephalothorax tissue. Thus, the CMNV RT-LAMP assay is a sensitive and specific new tool for the field detection and quantification of CMNV in the diagnosis and surveillance of covert mortality disease. © 2015.


Sun C.,CAS Qingdao Institute of Oceanology | Sun C.,National Laboratory for Marine Science and Technology
Physics of Fluids | Year: 2017

Quadratic flows have the unique property of uniform strain and are commonly used in turbulence modeling and hydrodynamic analysis. While previous applications focused on two-dimensional homogeneous fluid, this study examines the geometric structure of three-dimensional quadratic flows in stratified fluid by solving a steady-state pseudo-plane flow model. The complete set of exact solutions reveals that steady quadratic flows have an invariant conic type in the non-rotating frame and a non-rotatory vertical structure in the rotating frame. Three baroclinic solutions with vertically non-aligned formulation disprove an earlier conjecture. All elliptic and hyperbolic solutions, except for the inertial ones, exhibit vertical concentricity. The rich geometry of quadratic flows stands in contrast to the depleted geometry of high-degree polynomial flows. A paradox in the steady solutions of shallow-water reduced-gravity models is also explained. © 2017 Author(s).


Mo Z.,Chinese Academy of Fishery Sciences | Mo Z.,National Laboratory for Marine Science and Technology | Li S.,Ocean University of China | Kong F.,Ocean University of China | And 3 more authors.
Journal of Applied Phycology | Year: 2015

This study investigated a new fungal disease that attacks Pyropia yezoensis, an economically important red alga that is extensively cultured in China. An incidence was found in a P. yezoensis farm during mid to end November 2012 at Haizhou Bay, Jiangshu Province, China. Histopathology revealed that the naturally infected thalli were overwhelmed by a fungus, leading to progressive red rot symptoms. The causative agent was isolated, grown in pure culture, and identified as a member of the genus Alternaria by morphology and sequence analysis of the nuclear ribosomal DNA containing the internal transcribed spacer (ITS) region of ITS1 and ITS2. In artificial infection experiments on P. yezoensis blades, the fungal isolate was able to cause the same characteristic histopathology seen in natural infections. This fungal isolate grew well at a wide range of temperatures (8–36 °C) and at low salinities (5–50 ‰). In an orthogonal test used to determine the effects of environmental factors (temperature, salinity, and conidia concentration) on disease expansion, it was found that higher temperatures and lower salinities easily caused red rot disease, with the optimal conditions for disease development being 23 °C, 24 ‰ salinity, and a conidia concentration of 105 mL−1. This is the first report to show that Alternaria causes red rot disease in P. yezoensis. © 2015 Springer Science+Business Media Dordrecht


Liu P.-F.,Chinese Academy of Fishery Sciences | Liu P.-F.,Dalian Ocean University | Liu Q.-H.,Chinese Academy of Fishery Sciences | Liu Q.-H.,National Laboratory for Marine Science and Technology | And 3 more authors.
Journal of Invertebrate Pathology | Year: 2015

Thioredoxin (TRX), a major intracellular antioxidant, has a wide range of biological functions. It was up-regulated and targeted by WSSV. However, the relevance of TRX with WSSV infection and signaling pathway remains largely unknown. Sequence analysis indicated that TRX might interact with the WSSV030 (VP362) and WSSV454 (thymidine kinase-thymidylate kinase, TK-TMK) of WSSV. In this study, TRX, VP362 and TK-TMK were expressed and the interaction of TRX with VP362 or TK-TMK was investigated. Furthermore, how TRX affect the process of WSSV infection and the gene transcription of inhibitor of nuclear factor kappa-B kinase (IKK), a p38 mitogen-activated protein kinase (LvP38) and signal transducer and activator of transcription (STAT) in the hemocytes and hepatopancreas was explored. Far-western blot and enzyme-linked immuno assay (ELISA) results showed that TRX interacted with VP362 and TK-TMK. The mRNA expressions of IKK, LvP38 and STAT were significantly affected by the over-presence of TRX of Litopenaeus vannamei. Neutralization experiment in vivo indicated that TRX induced the transcription expression of VP28 and increased the viral copy numbers in the early stage of WSSV infection and it may attribute to the death of shrimps infected by WSSV. © 2015 Elsevier Inc.


Huang L.,CAS Qingdao Institute of Oceanology | Huang L.,University of Chinese Academy of Sciences | Huang L.,Qingdao University | Li G.,Chinese Academy of Fishery Sciences | And 8 more authors.
PLoS ONE | Year: 2015

Background: Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity. Methodology/Principal Findings: A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Conclusions/Significance The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder. © 2015 Huang et al.


Zhang Q.,Chinese Academy of Fishery Sciences | Zhang Q.,National Laboratory for Marine Science and Technology | Liu Q.,Chinese Academy of Fishery Sciences | Liu S.,Chinese Academy of Fishery Sciences | And 13 more authors.
Journal of General Virology | Year: 2014

A new nodavirus, named covert mortality nodavirus (CMNV), is associated with covert mortality disease of shrimp which has caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by reverse transcription (RT)-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in the hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in the muscle and lymphoid organ. The tubular epithelium of the hepatopancreas showed significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shared 54, 53 and 39% identity with the amino acid sequences of RNA-dependent RNA polymerase from Flock House virus, black beetle virus and MrNV. The results of fluorescence in situ hybridization showed that the hepatopancreas, striated muscle and lymphoid organ were positively reacting tissues. The mean size of negative-stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV, and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV-positive. © 2014 The Authors.


Yang H.-L.,Chinese Academy of Fishery Sciences | Yang H.-L.,Shanghai Ocean University | Yang H.-L.,National Laboratory for Marine Science and Technology | Huang J.,Chinese Academy of Fishery Sciences | And 9 more authors.
Letters in Applied Microbiology | Year: 2014

White spot syndrome virus (WSSV) is one of the main pathogens seriously threatening penaeid shrimp farming in the world. This study designed and synthesized five specific primers based on the conserved sequences of WSSV genome, optimized the reaction system and conditions, and finally established a cross-priming amplification (CPA)-based detection method for WSSV (WSSV-CPA). The results indicated that the optimized reaction temperature and time of WSSV-CPA were 63°C and 60 min, respectively. The detection limit of the WSSV-CPA assay was as low as 10 copies μl-1 and shared same sensitivity with the WSSV-qPCR assay. Due to dispensing with expensive thermal cycler, time- and cost-saving, and ease of use in field, it is anticipated that the WSSV-CPA method developed in this study will be instrumental for the diagnosis and surveillance of WSSV. © 2014 The Society for Applied Microbiology.


Liu Q.-H.,Chinese Academy of Fishery Sciences | Liu Q.-H.,National Laboratory for Marine Science and Technology | Ma F.-F.,Chinese Academy of Fishery Sciences | Ma F.-F.,Shanghai Ocean University | And 8 more authors.
Fish and Shellfish Immunology | Year: 2015

The interaction between viral structural proteins and host plays key functions in viral infection. In previous studies, most research have been undertaken to explore the interaction of envelope structural proteins with host molecules. However, how the nucleocapsid proteins of WSSV interacted with host molecules remained largely unknown. In this study, the interaction of nucleocapsid protein VP51 and ribosomal protein L7 of Litopenaeus vannamei (LvRPL7) was reported. Furthermore, the mRNA transcriptional response of LvRPL7 to WSSV was investigated. The results showed that LvRPL7 was widely distributed in all analyzed tissues of L. vannamei. The high expression levels of LvRPL7 were found in the tissues of muscle and gills. The temporal expression of LvRPL7 in WSSV-challenged shrimp showed that LvRPL7 was up-regulated (P<0.5) in the muscle at 8h and 24h post WSSV challenge and then restored to the normal levels. But the LvRPL7 expression was up-regulated (P<0.5) in the hepatopancreas at 8h post WSSV challenge and down-regulated at 12h and 24h post WSSV challenge. Indirect immunofluorescence assay indicated that LvRPL7 was mainly located on the surface and cytoplasm of hemocytes. Far-Western blotting showed that VP51 bound with LvRPL7. Moreover, ELISA results appeared that LvRPL7 interacted with VP51 in concentration dependent manner. Neutralization assay invivo showed that anti-LvRPL7 antibody significantly delayed WSSV infection. Our results reveal that LvRPL7 was involved in WSSV infection. © 2015 Elsevier Ltd.


Li J.,Chinese Academy of Fishery Sciences | Li J.,National Laboratory for Marine Science and Technology | Mo Z.,Chinese Academy of Fishery Sciences | Mo Z.,National Laboratory for Marine Science and Technology | And 5 more authors.
Fish and Shellfish Immunology | Year: 2015

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. The development of a live attenuated vaccine may be an effective approach for preventing this disease in fish. In this study, we introduced deletions of esrB, esaC, evpH, rpoS, and purA into the E.tarda LSE40δaroA strain, thereby generating five double-gene mutants (δaroAδesrB, δaroAδesaC, δaroAδrpoS, δaroAδevpH, and δaroAδpurA) and two triple-gene mutants (δaroAδesrBδevpH and δaroAδesaCδevpH). When blue gourami (Trichogaster trichopterus) was used as a fish model for the primary screening and evaluation of the vaccine candidates, all mutants were attenuated significantly by more than 2 to 3 logs in terms of the 50% lethal dose (LD50). Five double-gene mutants yielded relative percentage survival (RPS) rates of 26.1-82.6% after challenge with wild-type E.tarda. The δaroAδesrB mutant that conferred the highest RPS (82.6%) in blue gourami was also evaluated in flounder (Paralichthys olivaceus). After vaccination via intramuscular (i.m.) injection or immersion, this mutant could persist in the flounder for 14-35 days and it induced higher serum antibody titers than the control fish (P<0.01). Flounder vaccinated via i.m. injection at doses of 103-107CFU/fish had RPS rates of 14.3-66.7% after i.m. challenge with 104CFU/fish using wild-type E.tarda. Flounder vaccinated via immersion at a dose of 107CFU/ml exhibited 100% RPS against immersion challenge with 107CFU/ml using wild-type E.tarda. These results indicate that the δaroAδesrB mutant could be used as an effective live vaccine to combat edwardsiellosis in flounder. © 2015 .


Sun C.,CAS Qingdao Institute of Oceanology | Sun C.,National Laboratory for Marine Science and Technology | Ma X.,State Oceanic Administration
Science China Earth Sciences | Year: 2016

The vertical thermohaline structure in the western equatorial Pacific is examined with a Gravest Empirical Mode (GEM) diagnosis of in-situ mooring measurements. The poor GEM performance in estimating deep thermohaline variability from satellite altimetry confirms a lack of vertical coherence in the equatorial ocean. Mooring observation reveals layered equatorial water with phase difference up to 6 months between thermocline and sub-thermocline variations. The disjointed layers reflect weak geostrophy and resemble pancake structures in non-rotating stratified turbulence. A coherency theorem is then proved, stating that traditional stationary GEM represents in-phase coherent structure and can not describe vertically out-of-phase variability. The fact that stationary GEM holds both spatial and temporal coherence makes it a unique tool to diagnose vertical coherent structure in geophysical flows. The study also develops a non-stationary GEM projection that captures more than 40% of the thermohaline variance in the equatorial deep water. © 2016, Science China Press and Springer-Verlag Berlin Heidelberg.

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