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Immune Response of the Ridgetail White Prawn Exopalaemon carinicauda After Exposure to the Dinoflagellate prorocentrum minimum

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Journal of Ocean University of China 2023年第3期22卷 821-830页

作者： MU Cuimin REN Xianyun LI Jian Department of Aquaculture Research Laboratory College of Animal Science and Veterinary MedicineShandong Agricultural UniversityTaian 271018China Key Laboratory for Sustainable Utilization of Marine Fisheries Resources of Ministry of Agriculture Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdao 266071China Function Laboratory for Marine Fisheries Science and Food Production Processes Qingdao National Laboratory for Marine Science and TechnologyQingdao 266071China

The dinoflagellate prorocentrum minimum is known to affect the normal physiological function of Exopalaemon carinicauda by inducing oxidative stress,apoptosis,and cellular injury.To study the effects of P.minimum on the immune defense system of shrimp,E.carinicauda were exposed to 5×10^(3)cells mL^(-1)and 5×10^(4)cells mL^(-1)of P.minimum for 336 h in treatment groups,while E.carinicauda cultured in filtered seawater was employed as control.The total hemocyte counts(THC),hemocyanin concentration(HEM),and the activity of alkaline phosphatase(AKP)in hemolymph serum,as well as expressions of six immunity-related genes in hemocytes,hepatopancreases and gills were determined.The exposure of P.minimum significantly reduced the THC,HEM concentration and AKP activity in hemolymph serum.Immunity-related genes expressed differently in hemocytes,hepatopancreases and gills.Compared with the control group,the expressions of Crustin and pro PO in hemocytes were significantly up-regulated in the treatment groups,while the up-regulated expressions of LGBP,Lysozyme and Serpin were only found in the group exposed to 5×10^(4)cells mL^(-1)of P.minimum.In the gills of E.carinicauda exposed to P.minimum,the down-regulation of ALF,proPO and Serpin,up-regulation of LGBP and Lysozyme,as well as unaffected Crustin were observed.In hepatopancreases,the up-regulated expressions of LGBP,Crustin,Lysozyme,Serpin and proPO(only in 5×10^(3)cells mL^(-1)of P.minimum group)were found in the treatment groups.After exposure to P.minimum for 336 h,shrimps were injected with Vibrio parahaemolyticus and WSSV.The results showed that the mortality rates of shrimp in the treatment groups were significantly increased with a dose-dependent effect,which suggests that exposure to P.minimum may reduce the immunity of E.carinicauda.The research indicates that hemocytes and hepatopancreases play important roles in protecting the shrimp immune response to harmful algae,while the protection effect of hemolymph serum and gills may be suppressed.Since the exposure t

关键词： prorocentrum minimum Exopalaemon carinicauda immune response immunity-related genes

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Development of taxonomic rRNA-targeted probes of two harmful algae: prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

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Acta Oceanologica Sinica 2013年第2期32卷 66-75页

作者： CHEN Guofu WANG Quanfu ZHANG Chunyun ZHANG Baoyu WANG Guangce LU Douding XU Zhong YAN Peishen Harbin Institute of Technology (Weihai) The First Institute of Oceanography State Ocenic Administration Institute of Oceanology Chinese Academy of Sciences The Second Institute of Oceanography State Ocenic Administration

Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA （D1-D2） of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization （FISH） tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific （taxonomic）（Pmin0443 and Kmik0602） and the control probes （UniC0512 and UniR0499） were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence （p 〉0.05） was observed in the cell densities of the samples determined by FISH and light microscopy （LM）. All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

关键词： prorocentrum minimum Karenia mikimotoi fluorescence in situ hybridization taxonomic probe

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Identification of prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

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Acta Oceanologica Sinica 2009年第2期28卷 103-114页

作者： HOU Jianjun LAI Hongyan HUANG Bangqin CHEN Jixin College of Fisheries Huazhong Agricultural University Wuhan 430070 China College of Life Sciences Hubei Normal University Huangshi 435002 China State Key Laboratory of Marine Environmental Science Environmental Science Research Center Xiamen University Xiamen 361005 China Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology Yangtse River Fisheries Research Institute Jinzhou 434000 China State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China

Partial rDNA sequences of prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

关键词： oligonucleotide DNA probes prorocentrum minimum Takayama pulcheUa fluorescence in situ hybridization flow cytometry

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Competition of bloom-forming marine phytoplankton at low nutrient concentrations

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Journal of Environmental Sciences 2011年第4期23卷 656-663页

作者： Hanhua Hu Jun Zhang Weidong Chen State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China. School of Life Sciences Xiamen University Xiamen 361005 China Department of Gene Regulation and Drug Discovery Beckman Research Institute City of Hope National Medical Center Duarte CA 91010 USA

Competition of three bloom-forming marine phytoplankton （diatom Skeletonema costatum, and dinoflagellates prorocentrum minimum and Alexandrium tamarense） was studied through a series of multispecies cultures with different nitrate （NaNO 3 ） and phosphate （NaH 2 PO 4 ） levels and excess silicate to interpret red tide algae succession. S. costatum outgrew the other two dinoflagellates in nitrate and phosphate replete cultures with 10 μmol/L Na 2 SiO 3 . Under nitrate limited （8.82 μmol/L NaNO 3 ） conditions, the growth of S. costatum was also dominant when phosphate concentrations were from 3.6 to 108 μmol/L. Cell density of the two dinoflagellates only increased slightly, to less than 400 and 600 cells/mL, respectively. Cell density of S. costatum decreased with time before day 12, and then increased to 4000 cells/mL （1.5 mg/L dry biomass） at NaNO 3 concentrations between 88.2 and 882 μmol/L with limited phosphate （0.36 μmol/L NaH 2 PO 4 ） levels. In addition, P. minimum grew well with a maximal cell density of 1690–2100 cells/mL （0.5–0.6 mg/L dry biomass）. Although S. costatum initially grew fast, its cell density decreased quickly with time later in the growth phase and the two dinoflagellates were dominant under the nitrate-limited and high nitrate conditions with limited phosphate. These results indicated that the diatom was a poor competitor compared to the two dinoflagellates under limited phosphate; however, it grew well under limited nitrate when growth of the dinoflagellates was near detection limits.

关键词： Alexandrium tamarense nitrate phosphate prorocentrum minimum red tides Skeletonema costatum

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