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Abstract:In recent years, the research and construction of marine ranching in China have developed rapidly, which has promoted the development of marine ranching recreational fisheries and tourism industry. With the development of marine ranching recreational fishery, people need increasing recreational and ornamental tourism facilities, which requires more scientific ecological planning and management of the recreational marine ranching landscape. At present, the domestic recreational marine ranching mostly concentrates on fishing for the main leisure facilities, with fewer tourist ornamental landscapes and usually single types. Taking the marine ranching in Zhelin Bay as an example, the landscape ecological classification system of marine ranching in Zhelin Bay was established by applying the theory and method of landscape ecological classification. According to the similarity of appearance of features and related attributes, basic units were gathered into classes from the lowest level according to the top-down classification level principle of landscape ecological classification. The marine ranching in Zhelin Bay is divided into three first-level landscapes, five second-level landscapes, and sixteen third-level landscapes. The spatial analysis technique was applied to draw the landscape ecological classification diagram for each level, and the landscape distribution was discussed and analyzed. This study initially formed the marine ranching landscape ecological classification system, which provided the preliminary guidance and theoretical basis for further planning and research on the landscape pattern of marine ranching, and has important theoretical significance for improving the ecological and social benefits of marine ranching and promotion of the sustainable development of marine ranching. Promoting the upgrade of marine ranching to recreational fisheries and planning the construction of a visual ecological landscape could contribute to the construction of a marine ranching ecological civilization. Through the organic combination of the marine ranching natural environment, culture, and art, full use of the natural landscape resources and human landscape resources could be used and open up a new road for the development of marine ranching leisure fisheries and tourism.

Abstract:To acquire the actual wave force on an artificial reef under wave action and verify the stability of the artificial reef at the bottom of the sea, the influences of various nonlinear factors on the wave force acting on the artificial reef and numerical computation method for wave force should be analyzed and obtained. In this study, the nonlinear characteristic of the Morison equation for the horizontal wave force was analyzed using the dimensionless method based on the second-order Stokes wave theory. In addition, based on the computational fluid dynamics and finite volume method, adopting the Boundary wave method, a 3D numerical wave tank was established by the volume of fluid method. On account of the numerical results, the optimized relational expression of the maximum wave force and wave steepness was fitted by linear regression analysis method. The optimum equations of the maximum positive/negative horizontal force (Fmax/F–max) and wave steepness (δ) were Fmax = –0.89 + 110.44δ and F–max = –0.10–83.52δ, and the correlation coefficients were 0.9795 and 0.9899, respectively. The coefficient of anti-rolling and coefficient of anti-slide were 2.11 and 3.96, respectively, under the action of waves with the largest wave steepness. The results indicated that the main non-linear factor for the horizontal wave force acting on the artificial reef was wave steepness and the nonlinear effect enhances with increase in the number value. Taking a type of frustum cones artificial reef as an example, the maximum positive and negative horizontal wave force and the corresponding wave and pressure fields around the artificial reef were obtained. According to the numerical simulation results, the horizontal wave force of the artificial reef appeared to change periodically along with wave motion and increased with wave steepness. The results of stability calibration showed that the frustum cones artificial reef remained fairly stable on the sea floor at different test conditions, and it is well suited to the much larger wave heights in the sea.

Abstract:Based on survey data from Jiaozhou Bay in July 2017, the ecological characteristics of macrobenthos were analyzed using cluster analysis, and biodiversity and Multivariate-AZTI’s Marine Biotic Index (M-AMBI) methods. A total of 64 species were identified, including 25 species of Polychaeta, 23 species of crustaceans, nine species of mollusks, and seven species belonging to other benthic groups and echinoderms. The average biomass and abundance were 70.0 g/m2 and 132 ind./m2, respectively. There were three dominant species, including Ruditapes philippinarum, Nephthys oligobranchia, and Nemertinea. The ranges of the Margalef species richness index, Shannon-Wiener diversity index, Pielou’s evenness index, and M-AMBI were 0.69~3.77, 0.67~3.12, 0.25~1.00 and 0.26~0.60, respectively. According to the results of the cluster analysis, the macrobenthos at the 15 stations could be clustered into three infaunal communities at 15% similarity. The station communities with higher density of clam larvae were divided into the same community, clustering results were related to Philippine clam farming. The Shannon-Wiener Index (H′) and M-AMBI analysis showed that the macrobenthic community tended to be moderately disturbed.

Abstract:Large green tides of Ulva prolifera had become an annual event from 2007 to 2019 in the southern Yellow Sea and millions of tons of U. prolifera which were not salvaged settled to the bottom every year, releasing a large amount of biogenic elements such as C and N through degradation by microorganisms. This study discusses the effect of different temperature regimes on the release and the composition of dissolved organic matter (DOM) during the decomposition of U. prolifera. The results indicated that temperature significantly influenced the release of dissolved organic carbon (DOC) during 0~7 days (P<0.05): the DOC concentration at 20℃was significantly higher than that at 15℃ and 25℃. There was no significant difference in DOC concentration at different temperatures from day 7 to day 30 (P>0.05). For dissolved organic nitrogen (DON), temperature did not significantly affect the release of DON during the first 7 days (P>0.05). There were significant differences in DON concentration at different temperatures after 7 days (P<0.05): the concentration of DON was significantly lower at higher temperature (i.e. 25℃). The reason for this was that some organic nitrogen was converted to inorganic forms of nitrogen by microorganism during day 7 to day 30 and a higher microorganism abundance at 25℃ led to the transformation of more DON. The composition and fluorescence characteristics of fluorescent dissolved organic matter (FDOM) were evaluated by an excitation-emission matrix combined with parallel factor analysis (EEMs-PARAFAC). Three distinct dissolved organic matter fluorescent components (i.e. C1, C2, and C3) were identified using parallel factor analysis. C1, C2, and C3 were considered as tryptophan-like substances (also called protein-like substances), tryptophan-like substances and humic-like substances, respectively. The results showed that temperature had no significant impact on the composition of FDOM through comparing the relative content of three fluorescent components at different temperatures (P>0.05). While the fluorescence intensities of C2 and C3 were larger with the higher temperature (i.e. 25℃). Temperature affecting the fluorescence intensity markedly (P<0.05) may be caused by quicker microorganism decomposition rate under the higher temperature.

Abstract:Sagitta microstructure of young of the year Larimichthy polyactis collected in the Bohai Sea was studied, which is routinely used for age analyses. Results showed that there was usually one circular nucleus with one deeply black primordium in it, and only a few samples presented two nuclei or primordia. Average diameter of sagitta nucleus and primordia were (24.35±0.72) μm and (12.07± 0.58) μm, respectively. The first increment was observed on the second day, with a distance of (14.11± 1.08) μm to the central nucleus. The first feeding check appeared on the 4th increment, with a distance of (20.67±2.28) μm to the central nucleus, displaying larger width, deeper color, and higher clarity. From the otolith nucleus to approximately the 20th increment, increments gradually changed from a ring shape to an ellipse. Increment widths in the long axis ranged from 6.51 μm to 14.37 μm and tended to increase with age. Increment widths dramatically rose until approximately the 40th increment, reaching a value of (36.05±8.32) μm. Thereafter, increment mean width did not show any clear trend. The process of daily increment deposition of L. polyactis was accompanied by the formation of sub-daily increments. Sub-daily increment deposits displayed a lack of periodicity, irregularity, low clarity, and incompleteness compared with daily increments. Changing the depth of the field while observing these less defined increments resulted in a temporary elimination of the sub-daily increments from view. The analysis of otolith increment deposition has been widely applied and developed in age validation of larvae and juvenile fishes. The counting results directly determine analytical accuracy of the hatching date, spawning field distribution, and migration of wild individuals. The numerous presence of sub-daily increments will increase the difficulty of discriminating daily increments. L. polyactis has well-developed sagitta, which possesses greater width of daily increment and a greater number sub-daily increments. Therefore, distinguishing daily increments from sub-daily increments is the primary task of the research on the sagitta microstructure of L. polyactis. In the present study, the sagitta microstructure characteristics of L. polyactis were analyzed to provide a reference for daily increment judgment of wild L. polyactis and other fish species. Meanwhile, this study will supplement the biological information on L. polyactis and provide data for resource surveys of wild populations.

Abstract:When the loop-mediated isothermal amplification (LAMP) assay is used for detecting target genes, DNA extraction is unnecessary in many cases. Simple pretreatment (e.g. heating) is enough to obtain rather sensitive responses. Even test samples without any pretreatment can be used as template. This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies. In this study, using Stx1 gene from E. coli as model, we verified that viable cells, dead cells and extracellular DNA could function as template in the LAMP assay. In the incubation at 63℃, viable bacteria in the LAMP reaction mixture lysed completely within 2 min, providing DNA template for nucleic acid amplification. The Stx1 gene in diluted culture medium, spiked tap water, spiked seawater and real seawater all could be detected, with or without the step of DNA extraction. We found that the complex substances in real sample (e.g. natural seawater) exhibited considerable inhibitory effect on the sensitivity of the LAMP assay. These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring, bio-resource surveys, food safety, etc. in particular those based on environmental DNA.

Abstract:Ammonia can easily form in intensive culture systems due to the ammonification of uneaten food and animal excretions, and usually results in detrimental health effects to teleost. The aim of this study was to provide a reference value for the safe regulation and control of ammonia in the aquaculture of turbot, Scophthalmus maximus, and obtain the corresponding biomarkers of blood physiological indexes under acute ammonia stress. The acute toxicity effects of ammonia on young turbot [average body weight (163.90±15.31) g] were studied with a 96 h half-static toxicity test. The effects of different ammonia concentrations and exposure times on plasma physiological indexes were determined. The results showed that the semi-lethal concentration (LC50) of total ammonia (TAN) and non-ionic ammonia (NH3-N) for 96 h were 39.73 mg/L and 0.64 mg/L, respectively, when the sea water temperature was (19.0±0.5)℃, pH value 7.85, salinity 29.5, and dissolved oxygen (7.8±0.2) mg/L. The plasma epinephrine (EPI), cortisol (cortisol), superoxide dismutase (SOD), reduced glutathione (GSH), alkaline phosphatase (AKP), and blood glucose (GLU) contents or activities were significantly affected by both the ammonia concentration and exposure time. Furthermore, the interaction between ammonia concentration and exposure time also significantly impacted these indexes. The response time of plasma EPI, SOD, GSH, AKP, and GLU advanced with the increasing of ammonia concentration, while EPI, cortisol, AKP, and GLU showed a trend of increasing first and then decreasing with prolonged exposure. These plasma physiological indexes can be used as biomarkers for acute ammonia toxicity. The plasma physiological responses of the high concentration toxic group were characterized by their plasma SOD and GSH rapidly increasing over the exposure period (within 12 h), and GLU rapidly increased at 4 h and decreased sharply before death. The results suggested that the death of juveniles under acute ammonia toxicity may be due to oxidative stress, physiological metabolic disorders, and impaired respiratory function.

Abstract:To clarify the digestive and physiological characteristics in the early developmental stage of Seriola aureovittata, the activity of different digestive enzymes (lipase, amylase, trypsin, and alkaline phosphatase) were studied. Fertilized eggs and 1- to 60-day-old larvae and juveniles of S. aureovittata were selected as the research objects. The relationship between total length and days after hatching was also studied. The results indicated that the total length of larvae and juveniles increased with age within 60 days after hatching and increased significantly after 25 days. Lipase, amylase, and alkaline phosphatase were detectable during the embryonic stage while trypsin was detected in newly hatched larvae, which indicated that S. aureovittata can digest lipid, carbohydrate, and proteins before feeding. The specific activities of lipase and alkaline phosphatase increased rapidly after hatching (P<0.05), reached a peak at (5.88±0.45) and (5.56±0.41) U/mg, respectively at first feeding. The amylase activity reached its peak at (1.59±0.02) U/mg on day 7. The specific activity of trypsin increased slowly after hatching, and reached the highest value ((1098.67±24.03) U/mg on day 15 (P<0.05). Lipase, alkaline phosphatase, and trypsin were basically stable in the visceral mass of juvenile fish. However, the activity of lipase, alkaline phosphatase, and trypsin increased with the development of S. aureovittata. The amylase activity in the visceral mass of juvenile fish showed a downward trend and was basically stable at a lower specific activity level. The results of this study showed significant changes in various digestive enzyme activities during the development of larvae and juveniles, which were closely related to the development stage and feeding habits. The digestive enzymes were detected in early larvae that had not yet eaten food which was considered that the digestive enzymes were maternal in origin and not from exogenous food. The lipase, alkaline phosphatase, and trypsin activities in juveniles significantly increased, which reflect the improvement of intestinal structure and digestive function and the increasing demand for fat and protein.

Abstract:Protein kinase C (PKC) plays an important role in T and B lymphocyte-mediated immune response. However, the function of PKC is rarely reported in teleost fish. In this study, we have cloned the PKCα gene (namely CsPKCα), a typical member of the PKC family, and characterized its molecular features and expression pattern. CsPKCα cDNA is 2315 bp in length, with an open reading frame (ORF) of 2013 bp. qRT-PCR analysis showed that CsPKCα was expressed in various tissues, with a high transcription levels in the gill, liver, skin, and intestine, suggesting that it may play a role in immunity. Subsequently, we examined the expression of CsPKCα at different time points in six immune-related tissues (the gill, liver, skin, intestine, spleen, and kidney) after infection with Vibrio harveyi. CsPKCα was significantly upregulated in the gill, kidney, skin, and spleen at 24 h or 48 h post-infection (hpi) and downregulated in the liver at 12 hpi. The results suggested that PKCα might be involved in the immune response to V. harveyi; however, whether it is involved in macrophage activation during bacterial pathogen infection and the signaling pathways associated with T and B cells requires further investigation. This is the first report, to our knowledge, on the involvement of PKCα in the immune response to bacterial infection in fish.

Abstract:An understanding of the effects of aquatic environmental factors on elemental incorporation into otoliths is fundamental for reconstructing life and environmental histories of fish using otolith microchemistry analysis. This study investigated the effects of temperature (16℃, 19℃, and 22℃) and water elemental concentration (1×, 2×, 3× Sr ambient baseline, 6.5 mg/L) on the otolith elemental incorporation of Sr in larval-juvenile flounder, Paralichthys olivaceus (15~93 days post hatching). The results revealed that the otolith incorporation of Sr (Sr∶CaOtolith) was positively dependent on ambient Sr concentration (Sr∶CaWater) as well as water temperature. Sr∶CaOtolith values were significantly higher at 22℃ than at 16℃ and 19℃ at all Sr concentrations. Partition coefficient of Sr (DSr, Sr∶CaOtolith/Sr∶CaWater) tended to decrease with increasing Sr∶CaWater but remained stable when Sr∶CaWater reached a certain level at each temperature. Sr was incorporated into the otoliths more efficiently at 22℃ than at the lower temperatures. It appeared that the otolith elemental incorporation of Sr was closely related to the ambient elemental concentration and, thus, could reflect the water chemistry that fish experienced, suggesting that Sr could be used as an elemental fingerprint to reconstruct the life and environmental histories of the flounder during its early life stage in nature.

Abstract:The potential effects of starvation on the growth, feeding, and survival of Acanthopagrus schlegelii larvae were investigated at a water temperature of 18℃~19℃ by using ecological experimental methods. Along with the detection of the initial feeding rate and the point of no return (PNR), the optimum initial feeding time of the larvae were also ascertained. The results showed that larvae started feeding on the third day post hatching (dph), and the yolk sac and the oil globule disappeared at 4 DPH and 6 dph, respectively, which suggested that the duration of the mixotrophic stage was about 3 days and larvae were vulnerable to starvation. When the fish opened their mouths, the initial feeding rate was only 30%, which then rapidly increased to the highest level of 90% at 5 dph, after which it gradually declined to 45% at 7 dph, which denoted the PNR. Significant variation was found between the starved and control groups after 6 dph (P<0.05). Reduced mobility, distorted or deformed bodies, and increased mortality were observed in the starved larvae after the PNR. All the starved larvae died before 10 DPH. In brief, our study suggested that A. schlegelii larvae were vulnerable to starvation and the optimum initial feeding time of the larvae was within 4 days after their mouths opened.

Abstract:In this study, the karyotype and Ag-NORs of wild Hemibarbus labeo from Nandujiang River of Hainan were analyzed using the methods of intraperitoneal injection of phytohemagglutinin (PHA) and colchicine, and direct preparation of head kidney cells. Results showed that H. labeo had a diploid chromosome number of 50, the karyotype formula was 2n = 18m + 12sm + 12st + 8t, the number of chromosome arms (NF) was 80, no heterotypic chromosome was observed, and Ag-NORs were located on the ends of the short arms of pair No.5 and pair No.11. Comprehensive analyses of the chromosomal evolution in subfamily Gobioninae indicated that H. labeo had the primitive karyotype of Gobioninae fish. Our study will not only supplement the cytogenetic data of H. labeo and provide information on the taxonomy and evolution of the subfamily Gobioninae, genus Hemibarbus, but will also lay the theoretical foundation for germplasm protection and germplasm improvement of H. labeo.

Abstract:The spotted halibut (Verasper variegatus) is a rare and valuable marine fish species that inhabits the coast of northern China. Mature females are much larger than males because of their faster growth rate. It will create substantial economic benefits to establish an all-female breeding technique for V. variegatus. A better understanding of sex-related genes will contribute to the improvement of a single-sex breeding technique. In this study, we successfully isolated the piwil2 gene of V. variegatus, which was named Vvpiwil2. The total length was 3872 bp, including a 3192 bp open reading frame (ORF), encoding 1063 amino acids; the 5′UTR was 140 bp and the 3′UTR was 540 bp. Based on ExPASy, SMART, Signal4.1, and the NCBI Conservative Domain Database (CDD) biological analysis, the ORF encoded a putative protein, with a predicted molecular weight of 118.6 kDa and an isoelectric point of 9.02. No transmembrane structure or signal peptide site was detected. There were three domains: the ArgoL1, PAZ, and PIWI domains. Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of the Vvpiwil2 gene at different stages of embryo and larvae. The results showed that the Vvpiwil2 gene was abundantly expressed from early development to the high blastocyst stage, and then declined until the hatching stage. The developmental stage of the embryo from cleavage to the blastocyst stage was mainly guided by the cytoplasmic component. The mRNAs began to be transcribed and synthesized in a large amount at the early gastrula stage, then the transition from maternal to zygote occurred. Therefore, the results indicated that the Vvpiwil2 gene was a maternal gene. After hatching, the expression of the Vvpiwil2 gene at 68 days post hatching was significantly higher than during other stages, which demonstrated that the Vvpiwil2 gene was associated with gonadal differentiation. The expression level of the Vvpiwil2 gene in gonads was significantly higher than in other tissues, and the expression level in the ovary was significantly higher than in the testis, revealing that the Vvpiwil2 gene might play an important role in the maintenance of ovarian functions. The results of this study provide a potential sex determinant gene for V. variegatus and lay a solid theoretical foundation for the establishment of an all-female breeding technique.

Abstract:Cobia, Rachycentron canadum, the only species in the family Rachycentridae, is a candidate for cage culture in tropical and subtropical waters. Taiwan was the first to cage cobia in the early 1990s, and culturing of cobia has also been developed in Southeast Asia and other areas. Understanding the genetic diversity of cultured populations is important for the sustainable development and management of aquaculture. In the present study, 12 polymorphic microsatellite loci were selected to investigate and assess genetic diversity in five cultured populations of cobia from Beihai (BH), Lingshui (LS), Naozhou (NZ), Xuwen (XW), and Sanya (SY). As a result, 129 alleles were detected in the five populations. The mean number of alleles was between 3.833 and 6.750, the mean number of effective alleles ranged from 2.284 to 3.645, the mean of observed heterozygosity and expected heterozygosity was between 0.481 and 0.635, and 0.533 and 0.681, respectively, and the mean polymorphism information content ranged from 0.463 to 0.630. The population deviated significantly from a Hardy-Weinberg equilibrium at multiple microsatellite loci (P<0.05). Analysis of genetic differentiation indicated that the Fst range was from 0.055 to 0.150 and the genetic distance (D) range was from 0.240 to 0.635. BH and NZ had the highest Fst (0.150) and the highest D (0.635). The results of an analysis of molecular variance showed that 84% of the genetic variations were within cultured populations, and 16% were among cultured populations. A phylogenetic analysis using the unweighted pair group method with arithmetic mean and based on Nei’s genetic distance showed that one cluster comprising BH and SY, and the other cluster comprising LS and XW formed a branch, which was then clustered with NZ. These results provide data for further protection and improvement of the germplasm resources of cobia.

Abstract:Current shrimp farming industries face difficulties associated with disease outbreak caused by pathogens on shrimp larva. The application of probiotics may provide a solution to problems associated with disease. We used Pseudoalteromonas sp. strain KL-3 2010, which has antagonism against acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus (VPAHPND) and extracellular protease activity, and Exiguobacterium sp. strain KL-C2 2014, which has extracellular protease activity, as probiotics in a feeding test of Litopenaeus vannamei to investigate their effects on the growth and survival of shrimp. These two bacterial strains had no pathogenicity to L. vannamei in an immersion challenge at 108 CFU/ml. Weak positive signals for white spot syndrome (WSSV), VPAHPND, and Enterocytozoon hepatopenaei (EHP) were detected in the population of shrimp juveniles used in this study. L. vannamei was fed for 60 days with Pseudoalteromonas sp. KL-3 2010-supplemented diets (P group), Exiguobacterium sp. KL-C2 2014-supplemented diets (E group), alternation of the two diets (PE group), and normal diets (C group), respectively. Compared with the control group, the survival rate of the P group significantly improved (by 213%±43%; P<0.01), the average growth rate of E group significantly increased (by 105.5%±28.1%; P<0.05), and the survival rate and average growth rate of the PE group simultaneously improved (by 184%±52%; P<0.05 and by 70.6%±32.8%, respectively). Feeding with Pseudoalteromonas sp. KL-3 2010 and Exiguobacterium sp. KL-C2 2014 diets significantly affected the species of dominant microflora colonizing the shrimp gut, but these added probiotic bacterial strains were not detected in the re-isolated and cultivated dominant bacteria colonies. The addition with these probiotics to diets significantly increased some serum immune indexes, such as superoxide dismutase. The study showed that diets supplemented with the above probiotics may provide a practical approach for disease prevention and growth promotion in shrimp farming, even for shrimp positive for multiple pathogens.

Abstract:To explore the variations in zooplankton community in the shrimp factory aquaculture process, effectively manage water quality, and improve aquaculture efficiency, the characteristics and succession of zooplankton community structure, as well as the relationship between the community and Vibrio, phytoplankton, and the environment were analyzed. A total of 21 species, belonging to 4 categories were identified from August to November 2018. Protozoan zooplankton species were the most diverse, accounting for 61.9% of the total species, followed by Rotifera (14.3%), Copepoda (14.3%), and Cladocera (5%). The average density and biomass of zooplankton in the experimental pond was about 0.71×103 ind./L and 11.72 mg/L, respectively. The Shannon-Wiener diversity index (H') of the zooplankton fluctuated between 0.52 and 1.64. Correlation analysis showed that the zooplankton density was significantly negatively correlated with the phytoplankton density (P<0.05). Canonical correspondence analysis showed that temperature, pH, nutrients, etc. are important factors influencing the succession of dominant species of zooplankton. The results provide basic data for understanding the zooplankton community structure in the factory aquaculture of Litopenaeus vannamei.

Abstract:This study analyzed the effective growth, non-specific immune function, and water quality required for juvenile shrimps of Fenneropenaeus chinensis (initial weight, 0.08 g) at four stocking densities of G0 (250 ind./m3), G1 (500 ind./m3), G2 (1000 ind./m3), and G3 (2000 ind./m3) during a 40-d indoor culture experiment. The results showed that the survival rate and growth index of G1 and G2 at 20 d were not significantly different from those of G0 (P>0.05), while the survival rate and growth index of G3 was significantly lower than those of G0 (P<0.05). At 40 d, the growth index and survival rate of G1 was significantly lower than G0 (P<0.05) The growth index of G2 was significantly lower than G0 (P<0.05), while G2 the survival rate was extremely significant different from G0 (P<0.01). And the growth index and survival rate of G3 was extremely significant different from G0 (P<0.01). Measurement of the antioxidant status in the serum, hepatopancreas, gill, and muscle tissue showed that the total antioxidative capacity, superoxide dismutase, hydrogen peroxide, and glutathione peroxidase first increased and then decreased with increase in density. At 20 d, the four oxidation indices were significantly higher in G1 and G2 than in G0 (P<0.05), and at 40 d, the indices were significantly lower than those in G0 (P<0.05). The content of malondialdehyde (MDA) increased gradually with increase in density; the MDA of G3 was always significantly higher than that of G0 (P<0.05). The MDA of G1 and G2 was significantly higher than that of G0 in hepatopancreatic tissue at 40 d (P<0.05), and was not significantly different from that in the other tissues (P>0.05). The pH, DO, NO2-N, NO3-N, NH3-N, and COD had no significant effect on the main water quality indices (P>0.05) at different densities, which were all within the suitable range for the growth of F. chinensis. The results showed that density stress significantly affects the growth and antioxidant capacity of F. chinensis, and the suitable densities were 1000 ind./m3 for 20 d and 250 ind./m3 for 40 d.

Abstract:The identification of reference genes is critical for the establishment of sensitive and reproducible qRT-PCR-based assays. The current study was designed to explore the effects on acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains (VPAHPND) on the growth of shrimp in the presence of different concentrations of Sanguisorba officinalis L. alcoholic extracts and to select the optimal reference genes suitable for the evaluation of the inhibitory effect of S. officinalis L. on VPAHPND. The expression of six common candidate reference genes (rec A, pvs A, pvu A, gapdh, 16S rRNA, and rpo S) of VPAHPND under stress induced by S. officinalis L. alcoholic extracts were detected by qRT-PCR. Data analysis was conducted using the GeNorm, Norm Finder, Best Keeper, Delta CT, and Ref Finder software packages. The results showed that S. officinalis L. alcoholic extracts had a strong inhibitory effect on V. parahaemolyticus. The amplicons of these six genes had good specificity under the stress induced by different concentrations of S. officinalis L. extract. The lowest variation in Ct value was found for 16S rRNA (CV=3.88), and the highest variation occurred in pvs A (CV=12.53). The stability of the six reference genes judged by the five methods was as follows: The stability order results were: rpo S = 16S rRNA > gapdh > rec A > pvu A > pvs A from GeNorm; gapdh > rpo S > pvu A > 16S rRNA> rec A > pvs A from Norm Finder; 16S rRNA > rpo S > gapdh > rec A > pvu A > pvs A from Best Keeper; and gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A from Delta Ct. The comprehensive ranking result from by Ref Finder was rpo S > gapdh > 16S rRNA > pvu A >pvs A > rec A. After consideration of the pairwise variations, it is recommended to use both rpo S and gapdh as reference genes in these conditions. It was also revealed that the stability of reference genes differed between different strains and under different experimental conditions. With the improved experimental accuracy requirements, screening and verification of the appropriate reference gene has become an essential part of the experimental methodology. The results provide a foundation to support the study of the inhibitory mechanism of S. officinalis L. on VPAHPND through the perspective of gene expression. It is of great significance for the establishment of AHPND-prevention technology using S. officinalis L. as the core drug.

Abstract:Trehalose-6-phosphate synthase (TPS) is a key enzyme in trehalose synthesis and plays an important role in the response of organisms to stress. In this study, based on the unigene sequences from transcriptome sequencing of Litopenaeus vannamei challenged with high-temperature stress, the partial TPS cDNA sequence (complete ORF and partial UTR) (LvTPS) was obtained directly by PCR amplification. Sequence analysis revealed that the LvTPS sequence contained a 2529 bp open reading frame, encoding 842 amino acids with a molecular weight of 95.4 kDa and an isoelectric point of 6.17. LvTPS had two functional domains, the Glyco-transf-20 domain and the Trehalose_PPase domain. Multiple sequence alignments showed that LvTPS had the highest homology with the corresponding gene in Fenneropenaeus chinensis, with a similarity value of 63.73%. The phylogenetic tree indicated that LvTPS was closely related to that of F. chinensis, and clustered with invertebrates, such as Callinectes sapidus, Exopalaemon carinicauda, and Procambarus clarkia. Vertebrates were clustered onto a signal branch. Quantitative analysis of gene expression levels showed that LvTPS was widely expressed in all of the examined tissues with different expression levels. The gill and muscle had similar levels and the highest expression. The expression of the eye stalk, heart, and nerve was next, being significantly lower than that of gill and muscle (P<0.05). The expression in the hepatopancreas was the lowest, being significantly lower than that of the other five tissues (P<0.05). L. vannamei kept at 26℃ were used as control group. When the temperature was increased to 32℃, the LvTPS gene in the eye stalk and heart was significantly increased (P<0.05). When the temperature was increased to 38℃, the expression of the LvTPS gene significantly rose in gill, hepatopancreas, eye stalk, and heart (P<0.05). Expression in the hepatopancreas was significantly changed. Thereafter, the expression of the LvTPS gene decreased when the temperature decreased. When the temperature was returned to 32℃ and 26℃, the expression of the LvTPS gene in the six tissues was not significantly different from that of the control group. There was no significant difference in expression under different levels of temperature stress in the nerve and muscle. The above results indicated that LvTPS was closely related to the response to high-temperature stress. This study provided basic data for the analysis of the mechanism of the response to high-temperature stress in L. vannamei.

Abstract:Octopus ocellatus, affiliated to Octopodidae under the phylum Mollusca, is one of the most important economic species along the northern coastal areas of China. It has been considered as a candidate for aquaculture enhancement and release owing to its short life span and rapid growth. However, little is known about sexual selection before and after copulation in this species. To explore the mechanism of sexual selection in O. ocellatus, 11 morphological parameters of 36 adult octopuses were first measured and analyzed. Afterwards, 14 polymorphic microsatellite DNA markers were used to identify the paternity of 176 offspring and 3 female and 17 male parent candidates. The morphological parameters of male parents with and without offspring and those with high and low proportion of offspring were analyzed in three families by the t-test. The genetic similarity between female and male parents which had high and low percentage of offspring was also calculated. The results showed that there was a strong correlation between mantle length, wrist 2, wrist 6, and body weight in male octopuses, and that sexual dimorphism in the total weight and mantle width between male and female octopuses existed. Paternity testing revealed all three females mated with more than two males and 10 males mated with at least two females, which confirmed the polygyny and polyandry pattern in this species. There was no correlation between male octopuses with or without offspring and their morphological parameters. Moreover, the proportion of offspring was not related to the morphological parameters of males but was correlated to the genetic similarity and genetic distance between female and male parents—higher the genetic similarity, higher the proportion of offspring. This study provides a scientific basis for the resource protection and artificial breeding of O. ocellatus, and also, important information for research on the mechanism of sexual selection in marine cephalopods.

Abstract:As one of the main members in coral reef ecosystems, giant clams have important economic and ecological value. The conspicuous, brightly coloured mantle shared by all giant clams is a unique feature distinct from other bivalve mollusks and plays an important role in energy harvesting and privacy protection. The boring giant clam Tridacna crocea is usually found at depths of 0.5~3.5 m and is one of the most colourful giant clam species. In this study, T. crocea from Xisha, South China Sea was used to study mantle colour polymorphism by CSE-1 imaging and colour-measuring system and cluster analysis. Moreover, the relationship between mantle colour and morphological traits of T. crocea was also studied. The results showed that the mantle colour of most T. crocea individuals was distributed in the blue and red primary regions, and only a few individuals showed colour distribution in the green primary and bright green regions. T. crocea were clustered into six colour groups (including brown, reddish brown, light blue, dark blue, green and yellow), and the colour differences between each group were >15, among which the difference between dark blue colour of the fourth type and yellow colour of the sixth type was the largest (E=74.68), while the difference between light blue of the third type and dark blue of the fourth type was the smallest (E=18.98). The L, a, and b values of T. crocea mantle had significant positive correlations with shell height (P<0.05); the correlation coefficients were 0.2080, 0.2210, and 0.2375, respectively. This study provided a basic reference for the conservation of giant clam resources and the recovery of coral reef ecosystems.

Abstract:The envelope protein (ORF111) of Ostreid Herpesvirus 1 (OsHV-1) were studied using bioinformatics analysis, gene cloning and expression. Firstly, we designed the primers of the orf111 gene based on the complete genome sequence of OsHV-1, and successfully cloned the gene. We further analyzed the biological characteristics of the deduced protein sequence encoded by ORF111 gene, which including physicochemical properties, advanced structure, transmembrane region, and antigen determinant cluster. The results showed that the ORF111 protein is a stable hydrophobic protein, which contains nine antigenic determinants, five transmembrane domain structures, and a highly conservative pure ammonia arginyl-glycyl-aspartate (Arg-Gly-Asp, RGD) sequence. Then the OsHV-1-orf111 gene was linked with a pET28a(+) plasmid, and the recombinant plasmid pET28a-orf111 was successfully constructed. Finally, the recombinant plasmid was transformed into DH5α, and the recombinant protein was expressed after inducing by isopropyl-beta-D-thiogalactosine (IPTG). The SDS-PAGE test showed that the molecular weight of the ORF111 protein was about 32 kDa. In this study, an OsHV-1 envelope protein containing the RGD domain was successfully obtained by prokaryotic expression, which lays a foundation for the preparation of monoclonal antibodies of ORF111, and provides important basis for further study of the infection mechanism of OsHV-1. It also stimulates new ideas for the prevention and control of OsHV-1.

Abstract:To reduce the wastage of aquatic resources and improve the additional value of cod swim bladder, we determined the optimum technological conditions for antioxidant peptides from the cod swim bladder and observed the changes in antioxidant activities after being digested in vitro. Six kinds of proteases, namely, acid proteinase, dispase, pepsin, papain, compound proteinase, and trypsin, were used to hydrolyze the cod swim bladder in order to select the optimal protease by determining the degree of hydrolysis and DPPH radical scavenging rate. Combining the single factor method and response surface methodology, the optimum process was ensured at the hydrolysis temperature of 58.56℃, hydrolysis time of 6 h, pH of 7.21, and enzyme addition of 200 U/ml. The results of the confirmatory experiment showed that the scavenging rate of DPPH was 61.1%, which agreed with the predicted value of 62.0%, and the results proved that the optimized extraction process was stable and reliable. The free radical scavenging rates of peptides were detected, and the results showed that swim bladder peptides have good abilities of scavenging free radicals of DPPH, OH, O2-, and chelating Fe2+. Besides, the half maximal inhibitory concentration (IC50) of these was 8.29 mg/ml, 5.03 mg/ml, 5.43 mg/ml, and 1.35 mg/ml, respectively. After being digested in vitro, the degree of hydrolysis increased while the free radicals scavenging abilities and chelating power of Fe2+ decreased. This might have occurred because the changes of reaction conditions in the simulated gastrointestinal digestion process such as the change of pH value lead to the changes of composition and structure of the peptides. Meanwhile, the changes of composition and structure lead to the changes in antioxidant activities. In conclusion, swim bladder peptides had potent antioxidants and could serve as useful ingredients in the food industry.