Table of Content

25 April 2019, Volume 41 Issue 2

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Expression of of 3 beta-hydroxysteroid dehydrogenase gene (3β-HSD) in steroid genesis mechanism in the Fujian oyster, Crassostrea angulata

2019, 41(2):  87-95. 

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Crassostrea angulata is a major commercial species for the shellfish aquaculture in southern China. To investigate the reproductive endocrinology of mollusc，we studied genes encoding enzymes involved in steroid metabolism in C. angulata by molecular biology technology. A full - length cDNA (1 444 bp) encoding a peptide of 356 amino acids of 3-beta-hydroxysteroid dehydrogenase (3p - HSD) from C. angulata was obtained and the similarities of its deduced amino acid sequence to these of other molluscan species were analyzed. 3β-HSD mRNA transcripts was expressed in most tissues of oyster，at highest levels in gonad (P<0. 05 ) . The mRNA transcript of 3/3 - HSD increased from initiation stage to ripeness stages (P<0.05 )，and then the expression level decreased dramatically after partially spent ( P< 0. 05 ). The results elucidated the potential role of 3β- HSD in the steroid genesis mechanism of C. angulata. Furthermore, results obtained from in situ hybridization indicated that 3β- HSD mRNA signals were detected in the follicle cell of ovary. Combining the results of the in situ hybridization and qRT-PCR，it suggested that 3β - HSD might play an important role in the regulation of gonad development basing on steroido genesis mechanism in C. angulata

The purification and detection methods research of viral nervous necrosis in orange-spotted grouper Epinephelus coioides

2019, 41(2):  96-105. 

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Primers were designed based on various grouper nervous necrosis virus RNA2 genome sequence obtained from GeneBank. The coat protein gene of Fujian Xiamen Epinephelus coioides nervous necrosis virus was cloned and sequenced, while the sequence was submitted to the GenBank database under accession number MF510920. In the phylogenetic tree, the coat protein gene from XMNNV formed distinct group with that from RGNNV，but separated to the SJNNV, BFNNV and TPNNV nervous necrosis virus. Based on the result of molecular phylogenetic analysis, XMNNV was found belonged to red -spotted grouper nervous necrosis virus ( RGNNV) genotype. The virus particles were purified by ultracentrifugation.The results showed that it was an icosahedral virus with a mean diameter of 20?25 nm by electron microscopy, consistent with the reported structure of NNV. According to the sequence alignment results of XMNNV and the other RGNNV RNA2，the specific primers were designed according to the highly conservative sequence of capsid protein gene of viral nervous necrosis virus. A reverse - transcription polymerase chain reaction ( RT - PCR) assay for fish nervous necrosis virus was developed, the sensitivity of the method was 67 copies/jjlL. The isolated virus was used to infect E l l cell line and Larimichthys crocea muscle cells，and both cells showed sensitive for the isolate. While incubated with E l l cell, CPE was developed and vacuolation after incubation. L. crocea muscle cell was round and shedding off the wall, when broken up and died eventually. Besides, the infected samples were cloned and sequenced.The result of PCR indicated that the isolated virus was NNV. According to the transmission electron microscopy and PCR assay, we suspected the E. coioides was infected by red - spotted grouper nervous necrosis virus. This research provided effective method for the diagnosis and will be used in the field of prevention of grouper disease.

Analysis of oyster protein extracted by isoelectric precipitation

2019, 41(2):  106-112. 

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Using oyster as the raw material, the protein in oyster was classified by Osborne method, and the oyster protein was separated and extracted by isoelectric precipitation，while the molecular mass distribution was analyzed by SDS - PAGE. The results showed that the oyster protein component content was salt soluble protein > alkaline solution > water soluble protein > alcohol soluble protein. The optimal conditions for protein extraction were room temperature, alkaline solution conditions pH 12，1.0 h, acid precipitation conditions pH 4. 0 ，under which the protein extraction rate was 65. 6% and the purity was 84.3% ( dry basis) . SDS -PAGE electrophoresis showed that the water - soluble protein had obvious bands around 44.3 kDa and 14.3 kDa in the samples treated with p - mercaptoethanol；the salt soluble protein had obvious bands around 45 kDa and 20.1 kDa；alkaline solution appeared in three bands at 200, 44. 3 and 14. 3 kDa；and the protein sample prepared by the pH adjustment method showed a distinct band at 44. 3 kDa. In the samples without p- mercaptoethanol treatment，the salt soluble protein appeared in the band around 29. 0 kDa and 14. 3 kDa，while the protein sample prepared by the pH adjustment was at 97. 2 kDa. There were distinct protein bands.The research structure provides a theoretical basis for the further development and utilization of oyster protein.

Coastalline change and cause of formation in Huotongxi Estuary

Xiao-qin WU

2019, 41(2):  113-120. 

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Based on the google image data of different years, this paper made a comparative analysis of the shoreline changes in Huotongxi Estuary area using GIS technology. Combined with the numerical model analysis，it was considered that the reclamation of estuarine tidal flat wetland was the main reason for the change of shoreline in Huotongxi Estuary area. It is suggested that reasonable planning should be made in the future to reduce manual intervention on the shoreline.

Speciation and environmental risk assessment of heavy metals in surface sediment of Sansha Bay

2019, 41(2):  121-129. 

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BCR four stages sequential extraction procedure was applied to investigate the speciation and contents of six types of heavy metals ( including Cr, Co, N i, Cu, Zn and Pb) in the surface sediments in Sansha Bay. The relationships between the speciation of these heavy metals and other environmental factors were described. The environmental risk of heavy metals was evaluated by the ratio of secondary phase to primary phase ( RSP) and the contamination and potential ecological risk. The results showed that the Pb existed mainly in Fe - Mn oxide faction，and others existed mainly in residual fraction. Co, Ni，Cu, and Cr were significantly positively correlated with each other, and also with Pb and Zn. And the organic matter was significantly positively correlated with Co, Ni，Cu，Cr and Zn. The assessment results showed that Pb was generally at the middle pollution level, which might pose middle environmental risk, while other elements were at clean to light pollution level, whose environmental risk was low.

The spatial - temporal distribution of the nitrogen and phosphorus in the nearshore area of Fujian Province

2019, 41(2):  130-139. 

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The eutrophication of coastal waters has been the focus of domestic and foreign scholars. Based on the monitoring information of water quality in the nearshore area of Fujian Province during the different periods from 2015 to 2017, the spatial and temporal distribution of the nitrogen and phosphorus were researched. According to the law of spatial and temporal changes，the causes of eutrophication were also studied. Results indicated as follows：1) The water quality in the nearshore area of Fujian Province was generally good，and the concentrations of inorganic nitrogen and active phosphate in most areas reached the seawater quality standard. 2) The concentrations of inorganic nitrogen and active phosphate were higher in dry season than in wet season. 3) The concentrations of inorganic nitrogen and active phosphate in the sea area around Xiamen City and Ningde City were relatively high due to the typical topography and the human activities, such as fertilizer and manure use, fossil fuel consumption, and wastewater discharge. This research would provide scientific basis for the environmental management and protection in the nearshore area of Fujian Province.

Research on identification of major ecological risk sources in Dongshan Bay

2019, 41(2):  140-148. 

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In order to identify the main ecological risk sources in Dongshan Bay，a semi - quantitative risk identification method including the intensity, scope and frequency of ecological risk hazards was established. It could make a quick and accurate judgment of the main ecological risk sources and have a good assistant role in ecological risk assessment. Through identifying and analyzing 18 ecological risk sources involved in Dongshan Bay, it was indicated that reclamation, typhoon, nuclear power construction and operation, environmental pollution and biological invasion were the main ecological risk sources in Dongshan Bay. Identification and analysis of ecological risk sources can provide decision - making basis and theoretical support for regional ecological risk managers. In addition to strengthen the prevention of major ecological risk sources，Dongshan Bay ecological risk management needs to improve its ability to prevent rapidly growing risks in the long run.

Structural design of Wanshan artificial reef in Zhuhai

2019, 41(2):  149-156. 

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According to the hydrological and geological conditions of Wanshan sea area in Zhuhai, three kinds of artificial reef were designed. The reef was 3. 5 m×3. 5 m×3. 5 m frame box structure, and the frame adopts 0. 2 m×0. 2 m frame column. Under 25 years of wave conditions，the current force, anti -sliding stability, anti-overturning stability, bearing capacity of foundation and depression of the artificial reef were calculated. The results showed that when the bottom velocity was 1.2 m.s-1，the anti-sliding coefficient and anti-overturning coefficient of the reef were 2. 15 and 3. 69，the reef would not slide and overturn, and the bearing capacity of the foundation and depression met the requirements.

Acute toxicity of alizarin red S to Aspiorhynchus laticeps

2019, 41(2):  157-161. 

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In order to understand the acute toxicity of alizarin red S to Aspiorhynchus laticeps juvenile fish, the A. laticeps juvenile fish with body weight of(5. 8 ±0. 6)g was studied at 10℃ using a semi - static test method. The results showed that, when the concentration of alizarin red S was 400 mg/L, the mortality rate at 96 hours was 0；when the concentration of alizarin red S was 900 mg/L, all died within 72 hours；result showed the semilethal concentrations of alizarin red S on Aspiorhynchus laticeps at 24 hours,48 hours,72 hours, and 96 hours were respectively 788, 718, 651 and 618 mg/L. The safe mass concentration was 179 mg/L. Our results indicated that alizarin red S had low toxicity to Aspiorhynchus laticeps, which was suitable for tagging and releasing of Aspiorhynchus laticep. In the practical work, on the basis of the guarantee survival rate, our results suggested selecting concentrations between 179 mg/L and 400 mg/L as tags to get better marks.

Primary culture of grass carp brain cell in vitro

2019, 41(2):  162-166. 

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The establishment of in vitro cell culture system is an important approach in investigating the physiological metabolism. Meanwhile，the establishment of virus culture system based on stable cell is a necessary technique to isolate，identify and study fish viruses. As a kind of in vitro culture system, fish cell lines become significant experimental materials because of some advantages including low cost, high test repeatability and the experimental conditions can be controlled accurately. The cells in vitro from fish, as an significant research technology，has played a role in the theory and applied research of virology, immunology, fish resources protection，genetic breeding, oncology, environmental toxicology，fish physiology, endocrinology and so on. Although many fish cell lines have been established, relatively few are capable of viral propagation but producing the viruses slowly and giving low viral titers. In addition，its application is far less extensive than that of mammals. This study tried different methods to obtain grass carp primitive brain cells, including tissue block culture method, enzymatic digestion method and mechanical method. The brain cells collected by mechanical method were uniformly dispersed, and the cell formed cell monolayer after 24 h incubation in primary culture. The microscopy observation showed that cells were in polygon shape and adherent cells were mostly epithelial - like cells. The cell grew rapidly and was fairly homogenous in the L - 1 5 medium containing 10% fetal calf serum at the temperature 28℃. For mammalian cell culture,37℃ is usually chosen as the specified incubation temperature. However, fish culture cell temperature is different due to the differences among species. Fish are warm - blooded animals，which can maintain the integrity of cell function and physiology within a certain temperature range. Grass carp optimum growth temperature is 25 to 32℃, and the optimum culture temperature was consistent with the physiological and metabolic characteristics. MTT assay observed that the vitality of brain cells could maintain 3 days, and cells significantly degenerate after 3 days. The activity of cells on the 4th and 5th day was 11.7% and 34.8% lower than that on the 1st day, respectively, indicating that the 3d culture period is a suitable time for the in vitro experiment of primary brain cells of grass carp. The study indicates that the primary cells of grass carp brain can be used to analyze physiological mechanism in vitro. Fish cell culture techniques will improve future studies on viral infection，toxicological evaluation and the mechanism of nervous system.

Characteristics and application of chitin deacetylase

2019, 41(2):  167-174. 

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Chitin is a major component of shrimp and crab shells in fishery production. There are about 6 million to 8 million tons of discarded crab and shrimp shells produced each year. These discarded shrimp shells are usually dumped in landfills or discharged into the ocean, causing serious environmental pollution and waste of resources. Useful chemicals such as protein, calcium carbonate and chitin contained in shrimp and crab shells are ignored. Therefore, finding sustainable methods to make rational use of the crustacean shell has attracted widespread attention. The traditional method for obtaining chitin, chitosan and chitooligosaccharides is through the strong acid chemical digestion method. With the improvement of peopled environmental awareness, this method has been difficult to sustain. In addition, chitosan and chitosan oligosaccharides produced by chemical methods are difficult to control their degree of polymerization，degree of acetylation and acetylation mode, so it is difficult to study the relationship between the structure and function of chitooligosaccharides. Chitin deacetylase ( E. C. 3. 5. 1. 41) is an enzyme that can convert chitin oligosaccharides into chitosan oligosaccharides under mild conditions. Chitosan oligosaccharides with specific acetylation patterns have become an important research direction. Chitosan，a chitin deacetylated product, is a linear polymer composed of (3 - 1，4 - N - glucosamine, which is the only basic polysaccharide in nature. The partially acetylated chitooligosaccharide( COS) consists of N - acetylglucosamine ( GlcNAc, A ) and glucosamine ( GlcN, D ) and is a complex biopolymer with a variety of biological activities and potential applications. Chitosan has a broad spectrum of antibacterial properties, water retention，reproducibility，good biocompatibility, degradability, cell membrane permeability, high drug loading,non - toxic side effects,etc. Through glutaraldehyde after cross - linking, grafting, acetylation and other chemical and physical modification, it is widely used in medicine, environmental protection,food, agriculture,paper, cosmetics and other fields. Partially acetylated chitooligosaccharides are potent biological agents with anti - inflammatory, anti - tumor, immunomodulatory, neuroprotective and other important pharmaceutical activities. Their biological activities are thought to depend on their structure, ie their degree of polymerization and degree of acetylation, and their mode of acetylation. Chitin deacetylase (CDA) can directly deacetylate from chitin oligosaccharides to form chitosan oligosaccharides, which are widely present in eukaryotes and prokaryotes. The reaction conditions for the bio - enzymatic production of chitosan are mild, non - polluting,and the degree of polymerization，deacetylation and deacetylation of the product chitosan oligosaccharides are single, so chitin deacetylase has attracted wide attention. It has been reported that chitin deacetylase was successfully constructed into Pichia pastoris or Escherichia coli for heterologous expression. Chitin deacetylase (CDA) can directly deacetylate from chitosan oligosaccharides to form chitosan oligosaccharides, which are widely present in eukaryotes and prokaryotes. Several chitin deacetylases have been expressed in prokaryotic and eukaryotic hosts. The reaction conditions for the bio - enzymatic production of chitosan are m ild，non - polluting,and the structure of chitosan oligosaccharides are single, so chitin deacetylase has attracted wide attention. In view of the importance of this enzyme for industrial development, this paper reviews the sources of chitin deacetylase, deacetylation mode, catalytic mechanism and application of chitosan oligosaccharides.