Abstract:Hypoxia in the central areas of the Bohai Sea was reported for the first time in this study. The survey in August 2014 suggested that the hypoxic zone was 1200 km2 in area and the minimum DO concentration was 2.30 mg/L. The hypoxic zone was on the southwest side of the investigated area (119.1°–119.6°E, 38.3°–38.8°N). The DO (4 mg/L) isoline indicated that the hypoxic layer reached 10 m under the water. There were differences in the temperature and density between the surface and the bottom layer (δT and δρ), implying the formation of strong pycnocline in the hypoxic zone. The δT isoline of 5℃ mainly occurred in the vicinity of hypoxic zone with the highest δT value of 7.3℃. The density difference between the bottom and the surface layer has a similar spatial pattern with δT. By contrast, the salinity difference between the bottom and the surface layer was small (< 0.8) in the investigated areas. These results suggested that thermal stratification could be more influential than saline stratification in controlling the occurrence of hypoxia. The COD concentration was high (< 0.9 mg/L) on the southwest side of the investigated area that accounted for the majority of the hypoxic zone. In addition, high level of Chl-a (> 4 μg/L) and PO43--P (> 6 μg/L) was also observed in this area. We proposed that the formation of hypoxia could be a result of combined factors including the inflow from the Yellow Sea, the topography, and especially, the thermal stratification and in situ production.

Abstract:Studies on the species, abundance and distribution of zooplankton have been extensive and mainly focused on either the entire or certain small parts of the Bohai Sea. In this study, we analyzed the composition, abundance, dominant species, and biodiversity of zooplankton based on samples collected in the central Bohai Sea in 2013. The distribution and its influencing factors were explored with multivariate analysis according to the sampling date and environmental parameters. A total of 74 zooplankton species/taxa (including 21 pelagic larvae) were identified in four surveys. Copepods were the most abundant species. The numbers of pelagic copepod and medusa species accounted for 25.7% and 24.3% of the total species respectively. The composition of the dominant species was consistent with previous reports. The Calanus sinicus and Sagitta crassa were the dominant species. The abundance of C. sinicus and S. crassa explained 19.5%–50.3% and 7.3%–39.6% of the total zooplankton abundance respectively. In spring, the average abundance, the average biomass, the Shannon-Wiener index (H′), and the Margalef′s index (D) were 782.0 ind/m3, 157.1 mg/m3, 2.36, and 1.02 respectively. In summer, the values of parameters above were 199.6 ind/m3, 135.8 mg/m3, 1.75, and 1.78. In autumn, they were 42.1 ind/m3, 122.5 mg/m3, 1.83, and 2.08 respectively. In winter, they were 72.1 ind/m3, 151.1 mg/m3, 1.63, and 1.53 respectively. The abundance and biomass fluctuated and showed distinct heterogeneity in the central part of the Bohai Sea. There was a seasonal variation in the primary environmental factors that affected the distribution of zooplankton. In spring, they were surface salinity, bottom DO, and water depth. In summer, they were bottom temperature, bottom salinity, and chlorophyll. In autumn, they were surface temperature, surface pH, and bottom pH. In winter, they were bottom pH and chlorophyll. Our research provided the fundamental information on the long-term observation of zooplankton ecology in the central part of the Bohai Sea. Compared to the data collected in 1959, 1998, and 2006, the number of species, abundance and biomass of zooplankton have decreased.

Abstract:The community structure of phytoplankton could be affected by environmental factors, such as water temperature, salinity, total nitrogen, and total phosphorus. Changes in environmental factors may be associated with alteration in the community structure of phytoplankton. To investigate the consequences of oil spill in the Bohai Sea in 2011, here we analyzed features of phytoplankton community structure using data obtained in the comprehensive investigation at 41 stations within the central Bohai Sea in May, July, November and December 2013. The sampling and measuring methods followed the Specifications for Oceanographic Surveys and Specifications for Marine Monitoring. A total of 87 phytoplankton species were found, including 72 species of diatom in 33 genera and 15 species of pyrrophyta in 9 genera. There was another species of chrysophyta in 1 genus. Diatoms were the dominant phytoplankton species, while some dinoflagellates species also accounted for a large portion in the community. Compared to the historical data in the same season, there was an obvious shift in the community structure. Eucampia zodiacus, a previous dominant species, were not found in this survey; however, Thalassiosira punctigera appeared for the first time as a dominant species. The dominance of planktonic dinoflagellate became increasingly evident. The average cell abundance was 200.14×104 cell/m3, 16.32×104 cell/m3, 7.43×104 cell/m3 and 12.77×104 cell/m3 in May, July, November and December respectively. The cell abundance in May was higher compared to the historical data probably due to the outbreak of Guinardia delicatula, and in other month it was relatively stable. The diversity index and evenness index of the community structure followed the order July > November > May > December. The Spearman analysis revealed that changes in the nutrient structure of the central Bohai Sea was responsible for the shift in community structure, and that the spilled petroleum also might have affected the community structure. Because the phytoplankton community structure was generally consistent with results from other investigations, it was most likely unchanged after the oil spill in 2011.

Abstract:The Bohai Sea is a semi-closed gulf with weak hydrodynamic condition and self-purification capacity. Therefore its eco-system is more sensitive and fragile than open seas. A serious oil spill accident occurred in the Penglai 19-3 oil field located in the central Bohai Sea in 2011, which severely affected the marine ecological environment and fishery resources. To determine the aftermath in fishery ecological environment, nine surveys were carried out in the central Bohai Sea in 2012–2014. Based on part of the survey data, we analyzed the temporal and spatial variation in active phosphate and the corresponding factors in the central Bohai Sea. The results showed that the levels of active phosphate met the second class of sea water quality standard in spring and summer from 2012 to 2014, but the pollution caused by active phosphate was observed in part of the survey in autumn and winter; the horizontal and vertical distribution of active phosphate varied in different seasons, and in spring and summer the concentration of active phosphate gradually decreased along with the increase in depth, while in autumn and winter it was almost constant from the surface to the bottom; the seasonal mean concentration of active phosphate followed the order winter>autumn>spring>summer, and it was obviously higher in winter than in summer, plus the mean concentration was lower in 2014 than in 2013; The distribution and variation of active phosphate in the central Bohai Sea was affected by many factors, and key ones included exogenous supplement, endogenous release of nutrients, and biological consumption.

Abstract:In recent years, marine oil spill accidents occurred frequently, which has become a major concern on the marine environment and biological resources. To better understand the aftermath of an oil spill in a ship collision accident in the central Bohai Sea, here we modeled the floating and diffusion of oil film as well as the distribution of dissolved oil (petroleum hydrocarbon) using Euler-Lagrange method. The size of affected area and loss of fishery resources were also evaluated based on this model. We found that the volume of spilled oil was about 13,000 L, and the oil film lasted for 72 hours. The sea area affected by accumulated floating oil was about 69.19 km2. As the film broke down, oil droplets could be dissolved in the seawater in the form of emulsified and suspended particulate, and diffused to the coastal in 11 days, although the water quality would still meet the standard for fisheries. The oil film resulted in reduced fisheries resources, and the emulsified and suspended particulate could re-condense at the coast, which would harm the intertidal benthos. The affected area was determined based on the model and the length of coastline. It was estimated that the total economic loss in the oil film zone and the intertidal coastal zone was ¥ 6.319 million. Our study demonstrated that the numerical model, especially in the absence of observed data, could be a valuable tool in evaluating the change in natural fishery resources caused by oil spill accidents.

Abstract:The temporal and spatial distribution of chemical oxygen demand (COD) in different water layers, and its source and contribution to eutrophication were analyzed in this study based on data collected in the Bohai Sea in all 4 seasons of 2013. The results showed that the highest average concentration of COD appeared in summer, followed by autumn; while the lowest concentration appeared in spring. The analysis about vertical distribution of COD showed that the highest and lowest concentrations of COD appeared in the surface and middle layers in spring, and they appeared in the surface and bottom layers respectively in summer; in autumn and winter they were observed in the middle and bottom layers. As for the horizontal distribution the average COD concentration was distributed quite evenly in spring; in summer COD concentration was the highest in the western sea area and displayed a gradual decrease from the west to the east; in winter the highest concentration was detected in the northern area and it dropped from the north to the south. During the survey period, the COD level in most stations met the first-class seawater quality standard. The E value ranged from 0.088 to 2.995, and the average was 0.337±0.403. There were 25 stations in which the E values were above 1, including 20 stations in autumn and 5 stations in winter. These results indicated that the surveyed regions might not undergo eutrophication. The contribution of COD to eutrophication ranged from 46.15% to 141.41%, and the average was (71.36±14.98)%, which suggested that COD played an important role in the eutrophication of the Bohai Sea.

Abstract:To study the spatial distribution of mercury (Hg) in the central Bohai Sea, the surface sediment and the seawater were sampled in the survey in May of 2013. The result showed that the concentration of Hg in the sediment was (1.058–9.256)×10-3 mg/kg with an average of 4.781×10-3 mg/kg. In seawater it varied between 0.005 and 0.240 μg/L with a mean value of 0.090 μg/L. According to the national water quality standards (GB3097–1997) the concentration of Hg often failed the first (0.05 μg/L) and the second (0.20 μg/L) water quality standards, which demonstrated noticeable water pollution in the central Bohai Sea. There was no significant correlation between the Hg concentrations in the sediment and in the seawater at investigation stations (R=0.319, P<0.001, n=29). Grain size of solids was one of the most impactful factors that control the sedimentary variability of heavy metals; hence we normalized Hg concentration by the percentage of grains <20 μm. A significant correlation was then observed between the normalized Hg concentrations in the sediment and in the seawater (R=0.634, P<0.001, n=29). This suggested that Hg in the seawater came from the deposit Hg in the sediment. This “secondary pollution” in the sediment may cause long-term harm to the surroundings in the central Bohai Sea. Therefore we suggest policy makers should fully evaluate the environmental risks and bioavailability in the future economic activities in the central Bohai Sea.

Abstract:To study the temporal and spatial distribution of Mercury (Hg) and Arsenic (As) in the central Bohai Sea, we collected water samples of the surface, middle, and bottom layers in four survey cruises in May (spring), July (summer), November (autumn) and December (winter) in 2013. The results showed that the content of Hg was in the range of 0.0029–0.3926 μg/L with an average of 0.0676 μg/L. The vertical distribution of Hg concentration followed the order: bottom layer > middle layer > surface layer; the seasonal distribution showed the pattern: spring > winter > autumn > summer. Except for the surface layer in summer, Hg concentration in all water samples failed to meet the national water quality standard (GB3097–1997). The concentration of As ranged from 0.65 to 10.83 μg/L with the mean value of 1.50 μg/L, which met the requirement for the national water quality standard (GB3097–1997). The vertical distribution of As followed the order: surface layer > middle layer > bottom layer, and the seasonal distribution displayed an even pattern. The limit of As concentration is 20 μg/L according to the national water quality standards, thus the As level met the first water quality standard. However, the Hg level was readily higher than the first (0.05 μg/L) or second water quality national water quality standard (0.20 μg/L). These data suggested that the overall water quality of the central Bohai Sea was unsatisfying, and there was obvious pollution probably caused by human activities. Therefore the environmental risks in the Bohai Sea should be carefully evaluated in the future economic activities.

Abstract:In this study we investigated the changes and contributing factors of species composition, cell abundance, biological diversity and dominant species of phytoplankton community in the Liaodong Bay, with emphasis on their correlation with the 19-3 oil spill accident in Penglai. Our analysis was based on the field data about phytoplankton community collected in the Liaodong Bay in May (Spring), August (Summer) and October (Autumn) from 2012 to 2014. It was found the number of phytoplankton species was the lowest in May 2012, and the highest in October 2013. Overall the number in 2012 was lower than those in 2013 and 2014. The cell abundance of the phytoplankton community rose unexpectedly in all three summer seasons. The maximum reached 3072×104 ind/m3 in August 2012, and the minimum (27×104 ind/m3) appeared in May 2012. The dominant species in different seasons were nearly unchanged between 2012 and 2014. The dominant species in May were Paralia sulcata, Chaetoceros, Noctiluca scintillans, and it was Skeletonema costatum in August. In October Coscinodiscus, P. sulcata, and Chaetoceros gained the dominance. Interestingly, S. costatum, a dominant microalga, underwent explosive reproduction in August 2012, which might be associated with the increased oil concentration in surface seawater in the Liaodong Bay. Correlation analysis was performed to explore the links between the environmental factors and parameters such as species composition, cell abundance, and biological diversity index. The results indicated that the alteration in phytoplankton community structure was significantly correlated with water temperature, COD, dissolved oxygen and nutrients. Furthermore, the community structure was also strongly affected by the oil concentration in surface seawater.

Abstract:Ecosystems in the coastal water display high complexity and have been of great human and ecological interest. Interaction of physical, chemical and ecological factors determines the abundance and specific structures of biological communities, particularly the phytoplankton community, which comprise the lower levels of the oceanic food chain. To better understand the structure of the coastal phytoplankton community as well as its relationship with various environmental factors, a phytoplankton survey was carried out in the Laizhou Bay after the oil spills in Penglai 19-3 oilfield. At thirty-five selected sampling sites the water temperature, salinity, transparency, COD, pH, total nitrogen, and total phosphorus were investigated in May, August, and December from 2012 to 2014. Our sampling and testing methods followed the Specifications for Oceanographic Surveys and Specifications for Marine Monitoring. Correlation analysis (SPSS) was applied in determining the relationships between zooplankton communities and various environmental factors. Phytoplankton was collected using the standing net type Ⅲ (mesh size 76 μm, the standard sampling tool in Chinese marine phytoplankton studies) with a vertical haul at each grid station. Five classes including 125 species were commonly found in the surveyed area. Diatoms were the dominant species and dinoflagellates also shared importance in the phytoplankton community. Both the abundance and the diversity of phytoplankton reached the maximum in August, and the annual variation of diversity index and richness index tended to be consistent. The diversity of phytoplankton was positively correlated to the chemical oxygen consumption (COD, P<0.01) and negatively correlated to the transparency (P<0.01) in spring. There were negative correlations between the phytoplankton diversity and the surface temperature of seawater and inorganic nitrogen (P<0.01) in summer. As for the abundance of phytoplankton, it was negatively correlated to the depth of water, salinity, and dissolved oxygen (P<0.01) in autumn, and positively correlated to the petroleum content. These results showed that after the oil spilled, no obvious changes in the abundance and the community structure of the phytoplankton were present in the Laizhou Bay.

Abstract:In recent years, the marine environment and ecosystem in the Laizhou Bay have obviously deteriorated, which caused serious damage to the ecological environment and biological community. In order to understand the environmental qualities of the Laizhou Bay and the adjacent sea areas, we analyzed the distribution of salinity, pH, dissolved oxygen (DO), chemical oxygen demand (COD), disolved inorganic nitrogen (DIN) and PO4-P based on field data obtained from the Laizhou Bay in June 2013. The nutrition level and organic pollution in this area were also evaluated with potential eutrophication assessment standards and grading of organic pollution respectively. The results showed that DO and COD concentrations at all surveyed stations met the first class seawater quality standard. DIN pollution was serious mainly caused by the terrestrial input. At approximate 31% of investigated stations the DIN concentration exceeded the limit of the fourth class seawater quality standard. The level of PO4-P was lower and satisfied the first class seawater quality standard at all investigated stations. Our results suggested that the N/P ratio was higher than the Redfield value 16, and that phosphate was the limiting factor in the growth of phytoplankton. The nutritional pattern in the Laizhou Bay had evolved from nitrogen limiting to phosphorus limiting. The higher N/P ratio may alter the phytoplankton population, which will consequently affect the whole ecosystem. The nutrition status in the Laizhou Bay was phosphorus limiting potential eutrophication, and the organic pollution remained at the second level in spring 2013. These indicated organic pollution in this sea area; however, the pollution was alleviated compared to the summer 2007.

Abstract:In this study the hybridization between Epinehelus moara (♀) and Epinehelus lanceolatus (♂) were manipulated in the laboratory. The hybrid F1 larvae were hatched at salinity 30. The hatching and deformity rates of fertilized eggs and the survival activity index of newly hatched larvae was observed at salinities 5, 10, 15, 20, 25, 30, 35, 40, and 45. The growth and morphological characteristics of larvae, juveniles, and young fish was recorded. The results suggested that salinity 35–37 was optimum for the hatching of the hybrid F1, and salinity 20 to 30 was optimum for larval survival. SAI values of larvae became higher when the salinity was between 20 and 35, and they were lower when the salinity was 5, 10, and 45. The post embryonic development could be divided into the larval stage, the juvenile stage and the young fish stage, based on the features of the yolk-sac, the second dorsal fin spine, the pelvic fin spine, the scale and the body color. The embryo developed into pre-larvae in 2 days, and the average length of newly-hatched larvae was (1.959±0.152) mm. This stage was featured by yolk-sac and unabsorbed oil ball. The post-larvae stage lasted from Day 3 to Day 30, and the average length of 3-day larvae was (2.765±0.108) mm. At this stage the absolute length of the second dorsal fin spine and pelvic fin spine reached the maximum for larvae and juvenile fish. It entered into the juvenile stage starting from Day 31 after hatching and the average length was (18.130±1.565) mm. At this stage the visceral organs had been fully developed and the fish color became transparent. Starting from Day 46 after hatching it entered into the young fish stage when the average length was (39.850±2.565) mm and the body color turned light brown. At this stage brown spots appeared on the body surface and scales could be observed under microscope.

Abstract:In this study we investigated how mass selection would affect the genetic properties of the successive strains such as the fast growth in the shell width. Ten polymorphic microsatellite loci were analyzed to examine the genetic variation within a population, in one base stock, and in two successive mass selection lines of Pacific oyster (Crassostrea gigas). All microsatellite loci in the three groups showed high polymorphism, demonstrated by a large number of alleles per locus (NF0=16.5; NF1=12.2; NF2 =12.8) and high polymorphism information contents (Pic F0 = 0.9068, Pic F1 = 0.8982, Pic F2 = 0.8836). In all population-locus cases (3 populations × 10 loci), the observed heterozygosity values (Ho) of the 10 loci were lower than the expected values (He) (He=0.8954-0.9297, Ho=0.5775-0.6484). Twenty-four cases deviated from the Hardy-Weinberg equilibrium (P<0.05). The values of Fis ranged from 0.152 to 0.233, resulting in heterozygote deficiencies at the 10 loci in each population. Fst ranged from 0.0093 to 0.0245, indicating a weak genetic differentiation among the populations. The results suggested that the successive selection for rapid growing shell width might not reduce the genetic diversity. Therefore, the growth traits of C. gigas could be improved over generations under successive selection strains

Abstract:Because squid (Loligo japonica) is not one of the traditional food sources in many countries, systematic studies on its nutrition and frozen storage, even in countries where there is a long history of squid consumption, have been lacking. In this study the squids were frozen at –20℃, –30℃, and –50℃ for 90 days to investigate the change in the meat quality and stability under different temperatures. The quality was evaluated every 15 days during the frozen storage according to the sensory evaluation and parameters such as the total volatile basic (TVB-N), the myofibrillar protein content, Ca2+-ATPase activity, the sulfhydryl content, and thiobarbituric acid reactive substances (TBARS). The results showed that Ca2+-ATPase activity and mouth feel decreased with the prolonged time of frozen storage at different temperatures. In the first 15 days, decline in the Ca2+-ATPase activity of both samples was the fastest, and it slowed down afterward. The contents of myofibrillar protein and sulfhydryl was first slightly increased and then decreased. The values of TVB-N and TBARS were elevated in frozen storage at all temperatures; the higher the storage temperature, the more rapidly these values rose. The values of TVB-N and TBARS were increased at a much higher rate after about 45 days, which indicated the deterioration in meat quality. Microstructural analysis of squid showed that the muscle fiber bundles became loose during the frozen storage. The storage temperature significantly affected the rate of decline in squid quality. Storage at –30℃ and –50℃ better preserved the quality compared to –20℃. Therefore, –30℃ or below could be the recommended storage temperature.

Abstract:Here we studied the effects of four kinds of probiotics on the growth performance, intestinal flora, and resistance to diseases of GIFT strain of Nile tilapia (Oreochromis niloticus). Bacillus subtilis, Chinese herbal medicine compound Bacillus subtilis, compound probiotics, and Chinese herbal medicine compound probiotics were added into feed at the concentration of 2×107 CFU/g to form four treatment groups. The results were shown below: (1) All four probiotics obviously improved the weight gain rate of Nile tilapia (P<0.05). Probiotics also insignificantly promoted the survival rate and feed efficiency (P>0.05), and Chinese herbal medicine compound probiotics exhibited the best effect. (2) All four types of probiotics caused an increase in the total amounts of bacteria, bifidobacterium, lactobacillus, and bacillus in the guts of Nile tilapia (P<0.05), whereas the number of Escherichia coli was significantly reduced compared to the control group (P<0.05). These results indicated that all four probiotics in the feed could improve the structure of intestinal flora in Nile tilapia, and among them Chinese herbal medicine compound probiotics was the most effective. (3) After the Streptococcus agalactiae infection administered with artificial intraperitoneal injection, all the Nile tilapia in the control group died, but the infection was only fatal to some individuals in the treatment groups. Further tests confirmed streptococcus infection as the reason of death of Nile tilapia. The immune protection rates of the four probiotics were 51.42% (Group B), 58.62% (Group C), 58.62% (Group D) and 68.93% (Group E), again Chinese herbal medicine compound probiotics (Group E) showed the highest efficiency. In conclusion, adding a proper portion of Chinese herbal medicine compound probiotics into feed could effectively improve the growth index, the structure of intestinal flora, and the disease resistance of Nile tilapia.

Abstract:In the years of 2012 and 2013, there was an outbreak of disease and massive death of half-smooth tongue sole (Cynoglossus semilaevis) 15–20 days post-hatching (dph 15–20) in a hatchery of northern China, and the cumulative mortality reached 90%–100% within one week. The outbreak occurred in July and August when the water temperature was 22–24℃. The symptoms included erratic swimming behaviors such as spiraling movement and fast swimming in circles, without hemorrhaging and ulceration on body surfaces. The histopathological examination revealed typical signs of viral nervous necrosis. The nerve cells of brain and retina underwent severe vacuolation and necrosis. RT-PCR with primers of nervous necrosis virus showed positive results in all samples from moribund half-smooth tongue soles. The RT-PCR products were then sequenced and the sequence alignment was carried out with BLAST. It was found that the sequence similarity was above 98% between the nervous necrosis virus in half-smooth tongue sole (CsCN NNV) and five strains of red-spotted grouper nervous necrosis virus (RGNNV). In contrast, the similarities between CsCN NNV and other three genotype strains of NNV: Striped jack nervous necrosis virus (SJNNV), Barfin flounder nervous necrosis virus (BFNNV), and Tiger puffer nervous necrosis virus (TPNNV), were only 71%–78%. Therefore, we inferred that CsCN NNV was a new strain with RGNNV genotype. It was the first case of naturally occurred RGNNV infection of half-smooth tongue sole. Our findings provided insights into the epidemic mechanism of RGNNV infection as well as the prevention of viral nervous necrosis in half-smooth tongue sole.

Abstract:Natural resistance-associated macrophage protein (Nramp) belongs to the integration of membrane transport proteins, which has the capacity of enhancing macrophages that are meant to kill pathogens and innate resistance to intracellular parasites. In present study, cDNA of Nramp gene was amplified from spleen of half smooth tongue sole (Cynoglossus semilaevis) by SMART-RACE. The full-length cDNA of Nramp gene was 3717 bp, including 1677 bp open reading frame (ORF) encoding a protein with 558 amino acid residues, which contained the signature features of the Nramp protein family: 10 transmembrane (TM) domains, a consensus transport motif (CTM) with 20 amino acid residues. Compared with the other fish’s Nramp, C. semilaevis Nramp was the presence of one iron-responsive regulatory (IRE) protein-binding site in the terminal of ORF, which was similar to the vertebrate Nramp2. The deduced amino acid sequence of CsNramp exhibited about 63%−91% homology with 14 other vertebrate Nramp sequences. Phylogenetic analysis revealed that the CsNramp was clustered with other fish Nramp and was closer to Nramp2 of other species. RT-PCR results of the CsNramp transcripts in different tissues indicated that the CsNramp transcripts were highly abundant in spleen, kidney and low in muscle and gonad. The C. semilaevis challenged with the Vibrio harveyi could evidently elevate Nramp mRNA levels in spleen, kidney and liver, but the opposite phenomena were observed in the gills. To explore genetic variation and its relevant molecular markers in CsNramp gene, this research detected the polymorphisms of Nramp gene in one family of 233 individuals (68 infected individuals and 165 resistant individuals) by direct sequencing. Fifteen SNPs were detected in the partial of Nramp gene and 3 of them were genotyped successfully and SNP-g.3125(A→G) was significantly correlated to the resistance to Vibrio anguillarum. The results indicated that there were important effects on disease resistance of different Nramp genotypes, SNP-g.3125(A→G) can be used as potential genetic resistance marker loci, which can provide basic data for the genetic markers of C. semilaevis resistant breeding.

Abstract:Major fish viruses that are severely harmful in aquaculture industry include Lymphocystis disease virus (LCDV), Megalocytivirus (Mega), red-spotted grouper nervous necrosis virus (RGNNV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV) and infectious salmon anaemia virus (ISAV). Here we developed a specific amplicon rescue multiplex PCR (Arm-PCR) combined with gene microarray technique for the simultaneous detection of the seven types of fish viruses. First we optimized the conditions of Arm-PCR such as the annealing temperature and the concentrations of Taq DNA polymerase, Mg2+, dNTP and Primer Mix shown as follows. Reaction mixture (50 μl) consisted of 1.0 μl Taq DNA polymerase (2.5 U/μl), 5 μl 10×PCR Buffer (20 mmol/L Mg2+), 5 μl dNTP (2.5 mmol/L each), 9 μl 10×Primer Mix (2 μmol/L), and 1 μl template. The annealing temperature was 56℃. This method could simultaneously produce specific amplicons in one tube. The detection sensitivity of the Arm-PCR was 101 copies/μl for RGNNV, VHSV, non-structural protein of ISAV (ISAV-NS), and matrix protein of ISAV(ISAV-MA), 102 copies/μl for LCDV, Mega, IHNV, and IPNV, and 103 copies/μl for TRBIV (Turbot reddish body iridovirus). The Arm-PCR did not cause cross reactions with genomic DNA from healthy fish such as half smooth tongue sole, grouper, turbot and flounder.

Abstract:WSSV has been a globally recognized highly harmful pathogen in shrimp farming industry that causes tremendous economic loss. The envelope proteins of WSSV, VP28 and VP26, play important roles in interacting with host cells, initiating virus infection and mediating virus intrusion. In this study, we used pGAPZαA as the expression vector and X-33 Pichia pastoris as the host cell to express VP28 and VP26 in a secretive manner. The coding sequences of VP28 and VP26 (GenBank: AF332093.3) were amplified from WSSV using PCR, and the sequences of EcoR I (GAATTC) and Xba I (TCTAGA) were added to the 5¢ and 3¢ ends of the target genes. The purified PCR products were then cloned into the EcoR I/Xba I sites of the pGAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted into the reading frame. The construct was linearized by Bln I (Avr Ⅱ) and then was integrated into P. pastoris X-33 through electroporation while being screened by Zeocin. The expressed proteins were identified with SDS-PAGE. The VP28 and VP26 recombinant proteins could not be detected by coomassie brilliant blue R250 staining, however, the bands of the fusion proteins appeared after silver staining. The sizes of VP28 and VP26 fusion proteins were about 32 kDa. These results suggest that the P. pastoris system was effective in expressing WSSV envelope proteins VP28 and VP26, although the expression level was not sufficient. Nonetheless, our study still established a novel tool for the study of subunit vaccine, and provided basic information for the large scale vaccine production.

Abstract:White spot syndrome virus (WSSV) is one of the major pathogens that severely harm shrimp aquaculture. Different strains of WSSV display various virulence. In order to understand the geographic variation in fragments ORF14/15 and ORF23/24 of WSSV in China, we collected 48 samples of WSSV-infected shrimp from disease outbreak areas in 7 provinces of China between January and August in 2014. We identified the genotypes of WSSV-positive samples using PCR with ORF14/15 and ORF23/24 specific primers, and the amplified fragments were conjugated into T-vectors and transformed into the Top10 cells. We selected the positive clones and obtained their sequences. Then we compared the missing fragments of ORF14/15 and ORF23/24 from different samples with the sequences of TH-96-Ⅱ and China Taiwan strain (TW, AF440570) respectively. There were 21 samples with the products of ORF14/15 amplification, and 16 samples with the products of ORF23/24 amplification. The amplification ratios of ORF14/15 and ORF23/24 were 43.75% and 33.33% respectively. There were only 4 types of ORF14/15 compared to TH-96-Ⅱ. The lengths of the amplified fragments were 1260 bp, 1270 bp, 1892 bp and 2662 bp, corresponding to the missing 6540 bp, 6530 bp, 5908 bp and 5138 bp respectively compared to TH-96-Ⅱ. There were two types of ORF23/24 compared to the TW strain. The lengths of the amplified fragments were 1140 bp and 1146 bp, corresponding to the missing 12070 bp and 12064 bp respectively compared to the TW strain. These results suggested a certain degree of prevalence and variation of WSSV in China. There was an obvious difference in ORF14/15 between different strains，but only a minimal difference in ORF23/24. It is most likely that the variation in the missing fragments correlates with divergent WSSV virulence in different regions, and this notion needs to be further tested.

Abstract:Coat-epsilon protein (coat-ε) is a subunit of the coatomer complex that forms COPⅠ. To obtain the full-length sequence of coat-ε of Fenneropenaeus chinensis, we first acquired the sequences of 3' and 5' ends using rapid amplification of cDNA ends (RACE). The results were then spliced by DNAMAN to obtain the full-length 1402 bp sequence. The predicted 5' non-coding region (UTR) had 84 bp and the 3' non-coding region (UTR) had 310 bp. The open reading frame had 1008 bp that was supposed to encode 335 amino acids. The fragment including 230aa to 300aa belonged to the TPR superfamily. Signalp 3.0 server and TMHMM Server Version 2.0 analysis suggested that the amino acid sequence did not contain a signal peptide or a transmembrane structure. PSORTⅡ Prediction showed that coat-epsilon was probably located in mitochondria, cytoplasm, and endoplasmic reticulum. The phylogenetic tree analysis showed that coat-ε of F. chinensis was closely related to that of Arthropoda. We also analyzed the mRNA expression of coat-ε in different tissues with quantitative real-time PCR, and found that it was expressed in all tested tissues including appendage, hepatopancreas, epithelium, heart, stomach, intestine, eyestalk, muscles, gill, lymphoid organ and hemocytes. The expression level was the highest in muscles, followed by the gill and appendage. Our results provided important information for the functional study of coat-ε.