Abstract:Based on 245 samples collected in 19 cruises from 1960 to 2010 in adjoining waters of the Yellow River estuary, the long-term changes of the phytoplankton community and diversity were analyzed. These samples were towed from the seafloor to the surface at each site by using a standard phytoplankton net and subsequently checked for the species composition and cell abundance under an optical microscope in the laboratory. A total of 108 species were recorded, of which 87 were diatoms and 17 were dinoflagellates. spp. were the larger groups, with 27 and 10 species documented, respectively. The average total phytoplankton abundance during the last fifty years was 2.74×10⁶ ind/m³, with a peak of 16.8×10⁶ ind/m³ in the 1980s. Diatoms dominated the community in the last century, such as the large centric diatoms spp. However, the species in this century shifted to pennate diatoms like , as well as benthic species like . There was an increasing trend in abundance of the dinoflagellates spp. During the past fifty years, Coscinodiscus debilis, and were dominant in the community. However, the composition of dominant phytoplanktons had indeed changed and shifted. Under the co-influence of the absence of river flow and a strong El Niño event, the species abundances and diversity had decreased to the lowest levels during the last century, with average values only being 87.7×10⁴ ind/m³ (Shannon-Weaver index=1.97). During this century, however, the phytoplankton community and diversity in the Yellow River estuary have been altering under human activity, such as water-sediment regulation, with a steady recovery of 2.3 times and 16.8% increase over the levels at the end of the last century. This study on the long-term shift in phytoplankton community and diversity will provide a basic background for future protection of the estuarine ecosystem and enhancement of fishery resources in the adjoining waters of the Yellow River estuary.

Abstract:Zooplankton species play a crucial role in the pelagic food chain of coastal ecosystems and may reflect changes to marine environments. Therefore, community structure and seasonal variation in zooplankton assemblages are important for understanding ecosystem structure. In recent decades, anthropogenic activities have caused considerable changes to the environment of the Yellow River estuary and its adjacent waters. To ascertain how the present environment has influenced seasonal changes in zooplankton community structure, data on species composition, abundance, dominant species, and species diversity were collected during August 2013-May 2014 in the Yellow River estuary and its adjacent waters. Dominant species were selected based on a dominance() greater than 0.02. Between-season species fluctuations were described using seasonal replacement rate(). Species diversity in zooplankton communities were measured with the following indices:Margalef species richness('), and K-dominance curves. We identified 30 zooplankton species with 13 types of pelagic larvae, representing six phyla:Protozoa (one species), Chaetognatha (one species), Urochordata (one species), Ctenophora (one species), Coelenterata (eight species), and Crustacea (18 species). Seasonal replacement rates were 64.1% (summer to autumn), 71.4% (autumn to winter), 41.2% (winter to spring), and 70.6% (spring to summer), revealing obvious seasonality in species composition. Average zooplankton abundance was (2115.64±4143.70) ind/m³. Excluding , with Arthropoda accounting for 71.43%. Abundance was highest in spring (May), followed by winter (February), autumn (October), and finally, summer (August). Paracalanus parvus were the main dominant species, whereas was highly dominant in spring, exhibiting large blooms. All four species diversity indices were consistent in seasonal variation, being larger in summer and autumn than in winter and spring. Overall, zooplankton community structure was characterized by seasonal fluctuation. Copepoda and pelagic larvae were the dominant groups, with profound effect on variation in abundance and species diversity. Compared to previous research, zooplankton species composition and dominant species did not change significantly in the Yellow River estuary and its adjacent waters. The big blooms of were at least partially due to water organic pollution. Consequently, its increasing dominance underscores serious water eutrophication in the Yellow River estuary.

Abstract:Fishery-independent surveys are essential for collecting high-quality data to support the stock assessments and management of regular fisheries. In general, such programs are more costly and time-consuming than commercial fishery-dependent programs. Thus, considerable interest exists in using computer simulations to optimize methods for obtaining high-quality data with limited sampling effort. Currently, high intensity fishery-independent bottom trawl surveys may negatively affect and disturb fish populations and the ecosystem of fragile estuarine habitats. These areas support many important fishery species; however, they are also among the most extensively affected and threatened aquatic environments due to fishing pressure and environmental stressors such as coastal development. In this study, we developed computer simulations to evaluate and optimize sampling of mean body length and weight of target fish species in a cluster sampling survey. For use in simulations, bottom trawl surveys were conducted in the Yellow River estuary and its adjacent waters during 2013 (August, October) and 2014 (February, May) to collect abundance and biological-trait data on red tongue sole (). The relative estimation error (REE), relative bias (RB), and coefficient of variation (CV) were used to measure the performance (accuracy, precision, and efficiency) of sampling schemes. These indices increased for simulated data when the number of sampling sections decreased. In the current survey design, a reduction in sampling-section number from five to three would reduce sampling effort by 40%, while increasing REE by only~2% in about 40% of the catches. Thus, three sections are acceptable for surveys designed to obtain size-based indicators. This study also showed that sampling-effort optimization may vary between different survey objectives. Therefore, a post-survey analysis will improve fishery-independent survey designs based on specific survey goals, thereby yielding more effective survey data.

Abstract:Traditional fishery resources have declined in abundance or even been depleted in recent years as a result of multiple stress factors, including coastal development, marine environmental pollution, and high fishing pressure in the Yellow River estuary and its adjacent waters. has increased remarkably in abundance and been a dominant species in the Yellow River estuary and its adjacent waters, owing to its high adaptability, short life history, and high fecundity, as well as the decline in abundance of predators in these same waters. In order to ascertain the fishery and biological characteristics of to generate the basic parameters for its further stock assessment and management, the size composition, length-weight relationship, and sexual maturity of the species were examined based on the fishery and biological data collected in bottom trawl surveys conducted between June 2013 and May 2014 in the Yellow River estuary. The results showed that the standard length of the fish ranged between 25 and 154 mm, with a mean length of (87.77±24.181) mm. The dominant standard length class was 60-120 mm, accounting for 76.71% of the total abundance. The dominant class of body mass was 0.08-37.83 g, and the mean body mass was (9.1±6.905) g. The body mass class of 0-10 g dominated, occupying 63.03% of the total abundance of the species. The length-weight relationship with sex combined for the whole year was described as n=1842). Monthly changes in the sexual maturity periods and gonadosomatic index of were observed, where the reproductive period was from April to May and lasted to early June in the Yellow River estuary and its adjacent waters. There was no significant difference between sexes with regard to the relationship between percentage of sexual maturity and standard length of . A logistic curve was fitted to depict the proportion of sexual maturity in relation to standard length, and the parameters were derived from the curve, with a value of 0.039 and mean length at sexual maturity of 117.92 mm for the species in the Yellow River estuary.

Abstract:The index of biotic integrity (IBI) is sensitive to disturbance and can quantitatively describe the relationship between biological characteristics and the effects of human activities. It is one of the most widely used ecological indicators for evaluating aquatic ecosystem health. Originally, IBI was a type of water pollution assessment index that was established based on fish species composition, but it has now developed into a relatively complete evaluation system that has been used for the assessment of ecosystem health in bays, lakes, wetlands, forests, estuaries, and river systems. The comprehensive IBI index system based on fish has certain advantages over other evaluation indices because it includes information relating to fish population and individual status, as well as some aspects of community structure. Moreover, the IBI index system can be modified according to the objective and study area. In order to evaluate the biological integrity of the fish assemblage and the ecosystem health of the Yellow River estuary, bottom trawl survey data of fish species during 2013-2014 and historical data from the 1980s and 1990s were collected in the Yellow River estuary and its adjacent waters. A fish-based biotic integrity (F-IBI) index system for assessing habitat quality, which was originally developed by Karr, was proposed. The index system consists of 12 indicators that measure fish composition and richness, reproductive guild, and tolerance and trophic guild, taking into consideration the regional feature of fish species in Yellow River estuary. The evaluation criteria for F-IBI were also determined. Fish-based biotic integrity and habitat quality for the Yellow River estuary were evaluated. The results showed that the levels of fish biotic integrity in the Yellow River estuary during the 1980s, 1990s, and in 2013 were "excellent", "poor", and "very poor", respectively. Since the 1980s, the fish biotic integrity and environmental conditions have been declining in the Yellow River estuary. Anthropogenic activities such as overfishing have adversely affected the Yellow River estuary ecosystem and have led to the decrease or disappearance of fish species and decline of the ecosystem health of the Yellow River estuary.

Abstract:Water and sediment regulation involves artificially depositing sediments from rivers and reservoirs to the sea, thus increasing the main channel discharge capacity. Based on survey data before and after water and sediment regulation (June and July) in the Yellow River during 2011-2013, we used the index of relative importance (IRI), ANOVA, and multivariate analysis to evaluate the effect of this regulation procedure on fishery resources (species composition, distribution, and dominance) in the Yellow River estuary. We identified 92 species, including 52 fish species. Palaemon gravieri were dominant species, although their exact ranking varied across years. From 2011 to 2013, biomass first increased, then decreased, whereas fishery resources clearly changed in structure before and after water and sediment regulation. Average fishery resource density increased after water discharge and was low near the river mouth. CLUSTER analysis showed that between-year variation in community structure was the greatest, followed by variation resulting from water and sediment regulation, and finally, by variation between sampling sections. Post-regulation changes in water input and nutrient concentration of the Yellow River likely contribute to the variation in species composition and distribution in the region.

Abstract:is an important detritus-feeding fish and the prey of many high-trophic-level species in the Yellow River estuary ecosystem. Thus, has large effects on the abundance distribution of many fish species in the waters. The distribution of relative abundance in relation to spatiotemporal and environmental factors were analyzed using generalized additive models (GAM) based on bottom trawl surveys conducted during July, August, and October 2013 in the Yellow River estuary and its adjacent waters. Pearson correlation analysis was indispensable for determining whether factors introduced in the model were significantly correlated, as included variables should be independent. The relative abundance of varied noticeably with resource abundance, being high in autumn and low in summer. Additionally, was mainly distributed in the coastal waters of the northern Yellow River estuary; however, with seasonal decreases in food resources and temperature, the distribution gradually moved to the open sea. A minimum Akaike information criterion (AIC) was used to determine the best-fit GAM, via a stepwise method. Month, depth, sea-surface salinity, and phytoplankton abundance significantly affected the abundance distribution of in the Yellow River estuary and its adjacent waters. The final model explained 56.96% of the deviance, with phytoplankton abundance contributing the most (21.04%). abundance generally decreased with depth and increased with increasing phytoplankton abundance and sea-surface salinity. Specifically, abundance was relatively high in phytoplankton-rich waters <10 m in depth and with a sea-surface salinity of 17. These conditions may be related to reproduction-related migration, as well as estuarine environmental variation from the combined effects of freshwater runoff and tidal movement. In a future study, more data on quantifiable social and biological factors will increase the reliability of our conclusions. Nonetheless, the results of this study should contribute to the conservation and sustainable use of in the Yellow River estuary and adjacent waters.

Abstract:Interleukin-8 (IL-8), a chemokine that participates in the early inflammatory process, plays a key role in mediating resistance to infection in animals. However, there has been no information about IL-8 at the protein level for characterizing the regulatory role of this molecule during the immune response in fish. In this work, we constructed a prokaryotic expression system and prepared a polyclonal antibody against IL-8 of ). The coding region without a signal peptide sequence was first amplified and cloned into pET-32a(+), a prokaryotic expression plasmid. The recombinant plasmid was then transformed into Escherichia coli Rosetta. A soluble fusion protein was expressed after induction by isopropyl β-D-1-thiogalactopyranoside. The purified protein was detected as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a predicted molecular mass of 27.8 kD, suggesting that the prokaryotic expression system for IL-8 of crucian carp was successfully constructed. The purified recombinant protein was used as an immunogen to raise polyclonal antibodies in New Zealand rabbits. The serum antibody titer reached 1:105, as detected by indirect enzyme-linked immunosorbent assay. The antibody was purified by affinity chromatography and had a high binding activity and specificity for the recombinant protein antigen, as measured by Western Blot. Immunohistochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in crucian carp tissues. Comparing the results of immunohistochemistry with that of fluorogenic quantitative polymerase chain reaction, the expression of IL-8 was consistent between the protein and mRNA levels, and its expression ranked among tissues as follows:muscle > spleen > head kidney > intestine. This study provides a material basis for further research on the role of IL-8 in the immune response of .

Abstract:To investigate the functions of the capsular polysaccharide synthetic genes (GBS) isolated from Nile tilapia (cpsE and , were constructed by homologous recombination. Genomic DNA of GBS was used as a template to amplify the up and down homologous fragments of , whereas the pSET1 plasmid was used as a template to amplify the chromosomal chloramphenicol resistance gene ( and pSET4s-?/SUP> polymerase chain reaction (PCR) method. The recombinant plasmids pSET4s- were transformed into wild-type GBS by electroporation. Double-crossover and plasmid loss strains were obtained by changing the culture temperature. Finally, were screened for chloramphenicol resistance and the mutations were confirmed by PCR, real-time PCR, and DNA sequencing. To characterize , their growth rate, capsule thickness, capsular sialic acid content, and virulence were compared with those of wild-type GBS. The results showed that the growth rates of the wild-type, strains did not significantly differ. However, the capsule thickness, capsular sialic acid content, and virulence of were significantly lower than those of the wild-type strain. Further research suggested that is the critical synthetic gene of the capsular polysaccharide of GBS, whereas is important for capsular polysaccharide sialylation. The deletion of not only significantly reduced the capsular sialic acid content of GBS isolated from fish, but also significantly impaired its virulence.

Abstract:To study the role of heat shock protein 90 (HSP90) in the development of zebrafish embryos, we examined the mRNA levels of two HSP90 activity marker genes, namely (heat shock protein beta-1), at different developmental stages. Furthermore, we observed the marker gene expression and development of zebrafish embryos at different stages after treating with 2 μmol/L, 5 μmol/L, and 10 μmol/L of the HSP90 inhibitor radicicol. Analysis by quantitative polymerase chain reaction showed a strong increase in the mRNA levels of when zebrafish embryos were treated with radicicol at 12 or 24 hours post-fertilization (hpf). Western blot results showed that the protein level of HSP70 was upregulated when zebrafish embryos were treated with 5 μmol/L radicicol at 24 hpf, indicating that the activity of HSP90 was inhibited. Observations of embryonic development showed that the radicicol-treated embryos had a delayed development and survival rates of 95%, 77%, and 35% after treatment with 2 μmol/L, 5 μmol/L, and 10 μmol/L radicicol, respectively. The embryos manifested some abnormalities including reduced pigmentation, an enlarged pericardial membrane, and muscular dystrophy after treatment with 5 μmol/L radicicol at 72 hpf. The results showed that HSP90 plays an important role on the development of zebrafish embryos. The radicicol concentration of 5 μmol/L appeared optimal, because it strongly inhibited HSP90 without decreasing the survival rate of zebrafish embryos below 75%. Various morphological changes appeared in the radicicol-treated embryos, laying a foundation for the further study of HSP90's functions in zebrafish embryos.

Abstract:The purpose of this study was to observe whether dual-specificity phosphatase 1 () cells under cold stress. The recombinant plasmids plko.1--shRNA2, plko.1-negative control, and plko.1-EGFP were constructed using the restriction enzymes . These plasmids were separately transduced into 293T cells together with the lentiviral packaging vectors pCMV-DR8.9.1 and pCMV-VSVG. The virus packaged by 293T cells was used to infect the ZF4 cell line derived from zebrafish, and analysis by real-time polymerase chain reaction showed that knockdown ZF4 cell line, the cells were cultured at 28℃ (normal temperature control) or 10℃ (cold stress) for 10 days. Afterwards, they were stained with propidium iodide and annexin V-fluorescein isothiocyanate, then analyzed by flow cytometry. The results showed that the rate of apoptosis of the -knockdown cells increased significantly more under low temperature than that of the wild-type control cells did, as compared with the normal temperature control group. Furthermore, we designed two small hairpin RNAs named kd1 and kd2 based on the coding sequence of . Under cold stress, the frequencies of late and early apoptotic cells in the kd1-treated group were significantly higher, and the frequency of living cells was significantly lower, than those of the negative control group (<0.05). The cells cultured at 28℃ showed no significant difference in the rate of apoptosis according to silencing. However, under cold stress, apoptosis was significantly greater in the is involved in several physiological processes of fish, and it negatively regulates MAPK family proteins; for example, it inhibits p38 phosphorylation. This paper provides a new insight into the regulation of the apoptotic response of fish cells under low-temperature conditions by expression may relieve the inhibition of the MAPK p38 signaling pathway, which promotes ZF4 cell apoptosis under cold stress.

Abstract:Quantitative real-time PCR (qRT-PCR) is a powerful and commonly used method for in-depth analysis of gene expression that offers increased sensitivity and specificity over other methods. However, in order to obtain accurate results when using qRT-PCR to study gene expression, one or several internal control genes for normalization are needed. Housekeeping genes are known as such a class of genes that their expression levels are expected to remain constant in the cells or tissues in response to any environmental or physiological stress. But, in fact, no any housekeeping gene always stably expressed under all physiological conditions as ideal reference genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classic key glycolysis enzyme presented in all tissues, is one of the most common housekeeping genes used in the analysis of comparing gene expression levels as reference genes. Nowadays, the role of as the reference gene was being questioned and challenged by accumulated experiment evidences. To investigate the stability of as a reference gene, the full-length cDNA sequence was cloned from the ridgetail white prawn, , which mRNA was measured in different tissues and at different post-molt times. The obtained full-length cDNA of was 1514bp, including 69 bp of 5'-untranslated region (UTR), 1002 bp of open reading frame (ORF), 443 bp of 3'-UTR containing a canonical polyadenylation signal sequence AATAAA prior to a poly A tail. The ORF of encoded 333 amino acids without signal peptide analyzed by SignalP software which is highly conserved across the phylogenetic scale. The molecular mass was calculated to be 35.71 kDa, and the pI was estimated to be 6.61. By alignment, the amino acid sequence of binding domain (amino acids 3-149) and the catalytic domain (amino acids 154-311). In order to compare the expression stability of three endogenous candidate genes (18S rRNA, ) in qRT-PCR analysis in different tissues and different post-molt times, eight tissues (eyestalk, gill, heart, hepatopancreas, ovary, stomach, instestines and abdominal muscle) and 4 different post-molt times (1, 5, 10 and 15 min) of E. carinicauda were collected for qRT-PCR. Comprehensive analysis of the results using delta Ct method and the software packages geNorm, NormFinder and BestKeeper revealed that 18S rRNA was the most stable reference gene in both different tissues and different post-molt times, then was the -actin in decreasing order. In conclusion, the best choice for single reference gene is 18S rRNA, and 18S rRNA and E. carinicauda.

Abstract:This study aimed to obtain the genetic parameters of gynogenetic ENU-induced mutagenesis in grass carp (). The relative DNA content in grass carp with ENU-induced mutagenesis (Q group) and gynogenetic ENU-induced mutagenesis (E group) was 24.02 and 23.80, respectively. The Partec CyFlow ploidy analyzer determined that the values were close and that the mutants were diploid. We selected 28 microsatellite markers to investigate the genetic diversity of the two carp groups. Respectively, the E and Q groups had average alleles of 3.7143 and 5.1786, average effective alleles of 2.1857 and 4.0028, average expected homozygosity of 0.5122 and 0.2814, average expected heterozygosity of 0.4878 and 0.7186, and average polymorphism information content (PIC) of 0.4282 and 0.6606. The homozygous rate analysis of microsatellite loci indicated that the E group did not contain completely homozygous individuals, as all exhibited a degree of purity <1.00. The analysis also showed that homozygosity in several microsatellite loci (including , HLJC118) improved significantly at different rates. In summary, meiotic gynogenesis of ENU-induced mutagenic grass carp improved microsatellite homozygosity at different rates and significantly decreased genetic diversity. Thus, this method yields highly homozygous individuals that can provide important genetic data for improved selection of ENU-induced mutagenic grass carp.

Abstract:The Kumamoto oyster () was first identified in Ariake Bay, Kumamoto Prefecture, Japan, by Amemiya (1928) as a variety of the Japanese or Pacific oyster, ("shikame" means "wrinkled" in Japanese). Ahmed (1975) elevated it to species status () after careful morphological analyses. Banks et al. (1993, 1994) provided genetic support for (Amemiya 1928) as a species distinct from the closely related Pacific oyster C. gigas (Thunberg 1793). Wang and Guo (2008) developed a simple, fast, and reliable method using multiplex PCR to identify and C. gigas. The Kumamoto oyster is now known to be widely distributed across East Asia, including Japan (Ariake Sea and Seto Inland Sea), Korea (Suncheon Bay), and China (southern China). In Japan, Kumamoto oysters only command a small share of trade in the oyster culture industry, chiefly due to its small size. Therefore, little attention has been paid to securing aquaculture broodstocks except for a recent endeavor to produce hatchery seedlings. However, in southern China, recent sharp declines in wild resources due to environmental pollution and marine reclamation has led to the development of artificial to meet demands for increased oyster yield. Kumamoto oysters were introduced from the American Taylor Shellfish Farms as exotic germplasm. A core-based group was built following two methods, and the performance traits and introduction feasibility of the oysters were evaluated in comparison with Chinese stock as the control. The American stock has faster growth, lower viability, and higher commodity rate than the Chinese stock. After 1 year, shell heights and fresh weight of the American stock increased by 11.48% and 48.12%, respectively, compared with the Chinese stock. Stock source was the main factor affecting performance, and there was no significant effect of mating strategy on phenotypic traits. This study found that the American oyster in China has strong growth potential and provides valuable experimental material for improving the Kumamoto oyster, while supplying excellent variety for the oyster industry in southern China.

Abstract:The accurate estimation of additive and dominant genetic effects is fundamental to improving the accuracy of selective breeding and accelerating genetic gains. This study harvested 29523 individuals from 343 full-sib families (244 half-sib families) and examined the G7, G8, and G9 generations. The variance components of harvest body weight for four datasets (G7, G8, G9, and G8+G9) were estimated using average information restricted maximum likelihood. Two single-trait animal models were used for the analysis:(1) an additive genetic model comprising additive genetic effects plus common environmental effects (A+C), and (2) an additive-dominant model that includes dominant genetic effects (D) (A+D+C). For the A+C model, heritability estimates of harvest body weight for the four datasets were all low ( ≤ 0.15), ranging from 0.046 to 0.082. For the A+D+C model, heritability ranged 0.063-0.096, and the ratio of dominant genetic variance to phenotypic variance spanned 0.027 to 0.571. Harvest-body-weight heritability decreased in three datasets (G7, G9, and G8+G9), while increasing in G8. Low heritability estimates indicate that wild or improved populations with strong production performance must be introduced and integrated with the nucleus breeding population. Additionally, large between-dataset differences in the ratio of dominant genetic variance to phenotypic variance suggest that the accuracy of dominance variance estimates should be improved with new algorithms including more generations.

Abstract:To compare the growth differences between sea urchin hybrid families and evaluate the parents' combining ability in body weight, 42 hybrid families were produced. Using the restricted maximum likelihood and best linear unbiased prediction approaches, the breeding value of body weight at 250, 295, and 340 days of age, the general combining ability (GCA) of male and female parents and the specific combining ability (SCA) of parental combinations were evaluated. Differences in the phenotypic value and breeding value of body weight among families were compared using variance analysis. The results showed that both the phenotypic and breeding values of body weight significantly differed among families (<0.01). The breeding value of body weight showed a greater variation among families than the phenotypic value. When the proportion of the selected families was 50%, 88.89%-94.44% of the selected families were identical between the phenotypic and breeding value selection methods, and the breeding value selection method had a 0.93%-4.83% better selection efficiency. At the ages of 250, 295, and 340 days, the calculated GCAs of the male parents were -0.22-0.33, -0.31-0.41, and -0.29-0.31 whereas those of the female parents were -0.24-0.33, -0.31-0.41, and -0.28-0.28, respectively. The calculated SCAs of the parental combinations were -0.07-0.09, -0.10-0.13, and -0.32-0.32. At the ages of 250, 295, and 340 days, the male parents M5, M6, and M17 and the female parents F9, F11, and F19 always had higher GCAs than the other parents. The parental combinations M5F9 and M6F11 always had higher SCAs than the other combinations. The results provide a reference point for progeny testing and family selection in hybrid families.

Abstract:Groupers () are part of the large fish family Serranidae and are an important marine-cultured genus. In China, groupers have been farmed for over 30 years. Innovative technologies such as recirculating aquaculture systems (RAS) have been developed to improve the efficiency and sustainability of intensive aquaculture. In RAS, bacterial communities that are critical to ecological processes are also closely correlated with diseases in aquaculture. This study aimed to clarify bacterial community characteristics in marine RAS for groupers. Water samples were collected from the following pond types:fish-production, solid-liquid separation, sedimentation, biofilter, protein-separation, ultraviolet-sterilization, and oxygen-increasing. Samples were filtered through 0.22 μm mixed cellulose ester membranes to collect microbial biomass. Bacterial community DNA was isolated from the membrane filters. High-throughput sequencing (Illumina MiSeq) of the V3-V4 hypervariable region in the 16S rRNA gene was used to investigate microbiome structural characters. The results showed that Proteobacteria was the most abundant phylum in the water supply. Other important phyla included Bacteroidetes, Cyanobacteria, Fusobacteria, Firmicutes, and Nitrospira. Principal coordinate analysis and cluster analysis indicated that bacterial composition differed significantly before and after UV irradiation. The most dominant class in the rearing, solid-liquid separation, sedimentation, and protein-separation ponds was -Proteobacteria, whereas α-Proteobacteria was the most dominant in the ultraviolet-sterilization and oxygen-increasing ponds. Both classes were dominant in the biological-filter pond. The Shannon diversity index decreased first and then increased, peaking in the biological filter pond before decreasing again to the lowest value in the ultraviolet-sterilization pond, followed by a slight increase in the oxygen-increasing pond. In addition, our analysis of the water environmental parameters showed that dissolved oxygen concentrations increased, whereas NH₃ and NO₂^- concentrations decreased, remaining low after the water was purified. This outcome suggested that the RAS system has an effective water purification process. The correlations between microbial communities and environmental parameters were also investigated with canonical correspondence analysis, which showed that phosphates, pH, dissolved oxygen, and temperature had relatively stronger effects on bacterial communities than did other measured environmental variables. Overall, however, no significant association existed between environmental factors and microbial community structure, possibly due to small sample sizes.

Abstract:The present study was conducted to investigate the effects of exercise training on the growth, serum innate immunity, and hepatic antioxidant capacity of orange-spotted grouper (). The fish were exercised at an intensity of 0, 0.5, 1, and 2 body lengths per second (bl/s) for 8 weeks. Specific growth, weight gain, and survival increased significantly in the 1 bl/s group (<0.05). The plasma concentrations of total protein, globulin protein, complement 3, lysozyme, alkaline-phosphatase, and acid phosphatase increased as exercise intensity increased up to 1 bl/s and then decreased at the higher intensity of 2 bl/s (<0.05). The plasma contents of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase decreased as the exercise intensity increased up to 1 bl/s, and then increased at the higher intensity of 2 bl/s (<0.05). The albumin and complement 4 contents of plasma did not significantly differ among treatments. Hepatic total antioxidant capacity, catalase, and superoxide dismutase activity increased with exercise intensity up to 1 bl/s and then decreased at 2 bl/s (<0.05). The exercise treatments significantly affected the mRNA e xpression of HSPs70, and that of the 1 bl/s group was the highest. The results indicated that an exercise intensity of 1 bl/s effectively enhanced the growth, serum innate immunity, hepatic antioxidant capacity, and HSPs70 mRNA expression of .

Abstract:, which has rich agar, comprises the third-largest seaweed cultivation industry group in China, after . As light intensity gradually strengthens in summer, in the surface layer of cultivation areas becomes green, or even white, yet the internal part remains dark red. was cultured in artificial seawater with inorganic nitrogen (NO₃^-:NH₄⁺=2:1). Three conditions:low light low nitrogen (L-N-), high light low nitrogen (L+N-) and high light high nitrogen (L+N+) were settled in this study. The impact of light intensity and nitrogen on physiology and metabolism of was studied through measured matter accumulation, photosynthetic pigment, key enzyme during carbon and nitrogen metabolism under three conditions. The results showed that, compared with the L-N-group, the L+N-group showed decrease of soluble protein, water, phycoerythrin and chlorophyll-a, but the amount of soluble sugar increased 13.67%. Compared with the L+N-group, the L+N+ group showed the increase of soluble protein, water, phycoerythrin and chlorophyll-a, but the amount of soluble sugar decreased 16.3%. Compared with the L+N+ group, the L-N-group showed the increase of soluble protein, phycoerythrin, but there is no significant difference in its amount of water content, chlorophyll-a and soluble sugar. These results confirm that a sufficient amount of nitrogen can weaken the negative effects of high-intensity light on a red macroalgae.

Abstract:To characterize the changes in the protein content and tissue structure of the claw closer muscle of during the molt cycle, muscles harvested at various stages during the molt cycle were studied using paraffin section histology, electron microscopy, and biochemical methods. The results showed that the histology of the claw closer muscle changed noticeably during the molt cycle. The fibers and myofibrils were fully expanded in the inter-molt stage, and the cross-sectional area resembled an irregular circle or polygon with many nuclei under the sarcolemma. The sarcomeres were generally in register in this stage. During the later pre-molt stage, the cross-sectional area of fibers showed a wide size distribution owing to pro-ecdysial muscle atrophy, and many eroded areas appeared within the myofibrils. In the post-molt stage, the muscle fibers were compacting and shrinking. At the ends of the fibers, some sarcomeres were in a state of super-contraction, leading to the sarcomere shortening its resting length by up to 50%, whereas sarcomeres in the middle region of the fiber showed a "normal" appearance, with a typical arrangement of thick and thin filaments in the inter-molt stage. Biochemical experiments determined that the myofibrillar protein and soluble protein content in the claw closer muscle fluctuated and the results agreed well with the observed structural changes. The results indicate that the claw closer muscle of may increase in length during the molt cycle by adding new sarcomeres to the ends of the myofibrils. These new sarcomeres would begin as shorter sarcomeres when the carapace is very soft immediately after ecdysis. Then, as the carapace becomes hardened, the new exoskeleton would be stretched by the absorption of water and the super-contracted new sarcomeres would elongate to the length of the already-existing sarcomeres within the myofibril, resulting in muscle growth.

Abstract:The area of Haizhou Bay and its adjacent waters is an important spawning and feeding ground for many economically important fish and invertebrate species. It is also one of the important fishing grounds in the coastal waters of China. Environmental factors including sediment types, water depth, and hydrodynamics are heterogeneous in Haizhou Bay and its adjacent waters; this could affect the ichthyoplankton community structure during the main spawning period of spring and summer for many fish species. The species composition, dominant species, egg and larval abundance, and spatial structure of the ichthyoplankton community were analyzed using the index of relative importance and multivariate statistical analysis of data collected from fish eggs and larval surveys conducted by horizontal tows using plankton net, from May to July in 2015 in Haizhou Bay and its adjacent waters. A total of 33587 fish eggs and 713 larvae were collected during the surveys. Twenty-five species were identified from the eggs belonging to 25 genera and 14 families, while one species was unidentified. The fish eggs were dominated by Johnius belengerii, species. Thirteen species of larvae belonging to 13 genera and 13 families were identified, while one species was unidentified. The dominant species of larvae mainly included . Interannual variability of species composition of ichthyoplankton was observed compared to historical studies, and the main species were small fish species with short life cycles. Cluster analysis was used to divide the community structure based on spatial distribution of species composition and dominant species. The dominant species of ichthyoplankton varied depending on the months and transects. The abundance of fish eggs and larvae in nearshore waters was higher than that in offshore waters. The dominant species composition and abundance of ichthyoplankton varied between transects in May and June and was similar in two transects in July. The vertical profile of temperature and salinity of the two transects in Haizhou Bay and its adjacent waters, was used to analyze the relationship between the distribution of ichthyoplankton community and environmental factors. The spatial structure of ichthyoplankton community in the two transects varied during different months. Water depth, temperature, salinity, and other environmental factors had a comprehensive influence on the spatial variation of the ichthyoplankton community in Haizhou Bay and its adjacent waters from May to July. This study will provide a reference for the protection of the spawning ground, conservation of fishery resources, and management in Haizhou Bay and its adjacent waters. It is necessary to improve the protection of the spawning grounds in Haizhou Bay and its adjacent waters.

Abstract:Although dominant species exert the most control on community structure, the temporo-spatial heterogeneity of dominance as an integrative indicator has been often overlooked. To study fish community structure in Jiaozhou Bay, we analyzed 2008-2012 survey data from this area to determine species dominance (with IRI, index of relative importance) and variation in dominant species (with bootstrapping and multivariate linear regression). The results indicate that dominant species have changed substantially compared with previous studies. were the most dominant, comprising 9.79% and 10.71% of the total catch, with dominance indices of 3285 and 2068, respectively. Species dominance changed significantly between seasons, with in the summer, and in the fall and winter. Species composition differences were minor between the mouth and inner bay but notable in the outer bay. Multivariate linear regressions showed that during the survey period, decreased. This difference was significant across years ( < 0.01), implying obvious succession. Our analysis suggested that dominance heterogeneity between species may be driven by migration habits, seasonal fluctuations in water temperature, sediment type/depth, and the availability of forage organisms.

Abstract:Sharks occupy the top trophic level in the marine community and play an important role in maintaining ecosystem stability and diversity. The stock status of shark species is often difficult to assess by formal stock assessment methods due to limited fishery data. Blue shark () is the most widely distributed pelagic shark species in tropical and temperate oceanic waters. This species is often caught as bycatch in oceanic longline fisheries that target billfishes and tunas, and also in the artisanal longline fisheries that operate in coastal areas such as Chile. Because of its slow growth and late maturity, the blue shark is defined as "Near Threatened" globally in the IUCN species list. Determining the stock status of Indian Ocean blue shark using a data-poor approach has been assigned as a high research priority by the Indian Ocean Tuna Commission. In this study, we assessed the Indian Ocean blue shark stock status using the depletion-corrected average catch (DCAC) approach and Monte Carlo simulation. DCAC is a data-poor approach that only needs basic biological information (natural mortality, was estimated by the Hoeing method, resulting in a mean of 0.05. In addition to the annual catch data, the application of DCAC also needs the means and standard errors of the following parameters:depletion of the biomass (. First, we estimated the sustainable yield () of blue shark using abundance indices (standardized catch per unit effort[CPUE] time series) derived from different longline fleets (i.e., Spain, Portugal, Japan, and Taiwan, China). Second, we evaluated the sensitivity of DCAC by considering multiple combinations of different levels of , CPUE indices, and lengths of time series of data. Lastly, we summarized the estimated values and compared our estimates with the results from other assessment approaches for this species. The results showed that when F_MSY/M when was close to zero or negative. The results were sensitive to the CPUE index. The estimated was reliable and close to the maximum sustainable yield (estimated from other assessment models) when the Japanese longline CPUE index (1998-2014 or 2001-2014) was used. The current (2014) annual catch of blue shark might be at or just above the estimated maximum sustainable yield, although the estimate is subject to uncertainties. This study suggests that DCAC is suitable for estimating the of Indian Ocean blue shark using catch data and CPUE indices as the main sources. This study also provides guidelines for the application of data-poor approaches in domestic fisheries of China.

Abstract:This study aimed to assess the effect of aquaculture activity, especially the cultivation of large algae such as kelp, on the seasonal variation of and related parameters of the surface water were measured by the OceanPack_TM (SubCTech, Kiel, Germany) before and after kelp harvesting (May and August, 2015) in a typical polyculture area in Sanggou Bay, China. The variations of and its corresponding impact factors are discussed for different seasons and culture modes, and before and after kelp harvesting. The results showed that:(1) the mean values of in the surface seawater during the spring and summer seasons were (346.7±13.85) μatm(1 atm=101325 Pa) and (351.50±8.00) μatm in the inner bay, and those of the outer waters were (353.42±0.71) μatm and (358.05±2.01) μatm, respectively. All these values were lower than atmospheric decreased from the coast of the bay to the outside waters and rose at the reference outer region. The lowest values in both seasons appeared in the algal culture area and the highest values appeared in the shellfish culture area. (3) No significant correlation was found between CO₂ was significantly correlated with chlorophyll a and dissolved oxygen, indicating that organic activity had a great impact on and water temperature, dissolved inorganic carbon, chlorophyll a, and dissolved oxygen were significantly correlated. (4) The values of Sanggou Bay and the adjacent outer surface waters were lower than the atmospheric . The algal culture area was a strong carbon sink zone, because its value was much lower than that of the natural sea area. The shellfish culture area was a weak carbon sink zone, because its value was slightly higher than that of the natural sea area. The values in the algae-shellfish polyculture area were intermediate between the two monoculture areas. The photosynthetic activity of kelp in spring was a major factor affecting the surface water . The aquaculture activity in Sanggou Bay resulted in altered values compared with those of the natural sea area. The effects of physical factors on surface water became apparent in summer owing to a lack of aquaculture in that season.

Abstract:Chinese loaches (body weight:18-25 g) were exposed to 30 mmol/L NH₄Cl solution and air (exposure time:0, 6, 12, 24, 48, 72 h) to assess changes in tissue glutamine content, glutamine synthetase (GS) activity, and glutamate dehydrogenase (GDH) activity during ammonia loading and aerial exposure. Glutamine accumulation in liver and muscle, as well as GS activity in the brain, liver, and intestine, were observed with increasing duration of ammonia and aerial exposure. In all analyzed tissue except the liver, GDH activity was significantly affected by ammonia and aerial exposure. Our results suggested that Chinese loaches respond to internal ammonia increase through glutamine accumulation. Glutamine synthetase then stimulates the glutamine formation pathway and converts ammonia into non-toxic glutamine. The marked increase of GDH activity in the intestines demonstrated that intestinal GDH is more important than intestinal GS in the overall defense against ammonia toxicity. Across the exposure period, variation in liver GDH activity was not affected by ammonia and air, nor associated with a concomitant increase in GS activity and glutamine content that probably occurred due to efficient glutamine replenishment via transaminase action.

Abstract:To explore the toxic effects of several heavy metals on hemocytes from , we evaluated the cell viability (CV) and esterase activity (EA) of the -3 mol/L heavy metals (Cd²⁺, Hg²⁺, Cu²⁺, and Zn²⁺, compared with a control group without exposure to heavy metals) for 6 h by flow cytometry. The results showed no significant effects of 10^-9-10^-6 mol/L Cd²⁺ on CV and EA. 10^-5 mol/L Cd²⁺ significantly inhibited EA. Both CV and EA significantly decreased after exposure to 10^-4 mol/L and 10^-3 mol/L Cd²⁺. There were no significant effects of 10^-9-10^-6 mol/L Hg²⁺ on CV and EA, whereas 10^-4 mol/L and 10^-3 mol/L Hg²⁺ significantly decreased CV and EA. Concentrations of 10^-9-10^-5 mol/L Cu²⁺ had no significant effects on CV and EA; however, 10^-4-10^-3 mol/L Cu²⁺ significantly decreased CV and EA. Although 10^-9-10^-5 mol/L Zn²⁺ had no significant effects on CV and EA, 10^-4 mol/L Zn²⁺ significantly decreased EA, whereas 10^-3 mol/L Zn²⁺ significantly decreased CV and EA. The toxic critical concentrations of Cd²⁺, Hg²⁺, Cu²⁺, and Zn²⁺ were 10^-4 mol/L, 10^-5 mol/L, 10^-4 mol/L, and 10^-3 mol/L, respectively. The toxicity of these four heavy metals on > Cd²⁺ > Cu²⁺ > Zn²⁺. The toxicity of heavy metals on hemocytes was dose-dependent. EA is more sensitive to heavy metal stress, and could be used as an indicator for toxicology studies.

Abstract:Seahorses ( spp.) are an iconic and ecologically important species worldwide. Within the past decade, overfishing and habitat destruction has drastically reduced wild seahorse populations. Seahorse aquaculture was thus developed as an important strategy to alleviate pressure on wild stock; however, these farms have been severely afflicted with tail rot, a serious disease in seahorses. To identify the agent causing tail-rot disease in juvenile lined seahorses (), this study characterized the pathogen's physicochemical properties, extracellular product (ECP) activity, and drug susceptibility. One bacterial strain with superior growth (HM-10) was isolated from cultured liver samples of diseased juvenile lined seahorses. Healthy juvenile lined seahorses were then challenged with the strain through intraperitoneal injection. The results confirmed that HM-10 was pathogenic, causing tail-rot symptoms in challenged seahorses. The median lethal dose ( cfu/g body weight. Results from the API 20E identification system showed that under L-arabinose and amygdalin-derived acid production, arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase activity in HM-10 were negative, positive, and positive, respectively. Morphological observation, biochemical characteristics, and genetic analysis identified HM-10 as . The genetic analysis involved sequence detection of housekeeping gene 16S rRNA, including DNA gyrase subunit B gene (), DNA recombination protein gene (gyrB--recA. The ECPs of HM-10 were extracted and their properties analyzed. The results showed that the ECPs had clear hemolytic activity in response to sheep red blood cells, while also exhibiting protease, gelatinase, urease, amylase, and hemolytic activity, but not lipase or lecithin activity. Susceptibility experiments involving 33 drugs indicated that HM-10 was highly sensitive to 17:cefepime, ceftriaxone, cefotaxime, ceftazidime, cefepime, cefepime, cefoxitin, cefoperazone, kanamycin, spectinomycin, tobramycin, piperacillin, ampicillin, streptomycin, nitrofurantoin, chloramphenicol, and polymyxin B. In conclusion, the HM-10 strain of was the bacterial pathogen causing tail rot in lined seahorses. Our results are useful as a reference for the prevention and control of vibriosis in cultured seahorses.

Abstract:Spring viremia of carp virus (SVCV) was routinely isolated and identified from tissues of carp () from 40 carp farms in Heilongjiang Province by separation in cell culture and identification using polymerase chain reaction. SVCV isolates were subjected to virus titer determination, phylogenetic reconstruction based on their glycoprotein amino acid sequences and genotyping. Cell culture results showed that the isolates from four different farms infected carp epithelial cells and caused a cytopathic effect. The isolated SVCV strains were designated as Shlj1-Shlj4 and were confirmed as SVCVs by polymerase chain reaction. The titers of SVCV Shlj1-Shlj 4 were 10^6.28, 10^6.88, 10^7.57, and 10^6.38×the 50% tissue culture infectious dose/mL, respectively. The results of the Shlj glycoprotein gene cluster analysis showed that Shlj1-Shlj4 clustered with China strains A2 and BJ0505-2 as well as America strains USA and 212364. The nucleotide sequence similarities of Shlj 1-Shlj 4 with those previously reported SVCV strains were 98.4%-99.8%, and the similarities among Shlj 1-Shlj 4 were 98.6%-99.8%. The highest nucleotide sequence similarity (99.8%) was observed between Shlj3 and USA/212364, and Shlj2 had the lowest similarity (88.0%) with England reference strain 880163. The amino acid sequence alignment results showed that, among the four Shlj strains, the glycoproteins of Shlj 4 contained the most mutations. Genotype analysis showed that Shlj 1-Shlj 4 all belonged to genotype Ia. The study showed that the SVCV detection rate was about 10% in Heilongjiang Province during 2015-2016, and the glycoprotein gene sequences of SVCV strains from different farms varied, indicating that SVCV strains are evolving in the Chinese fish farm environment.

Abstract:Cold stress can cause metabolic disorders or death in aquatic organisms through severe effects on physiological and biochemical processes. The present paper reviewed the effects, mechanism, and control of the mechanisms of cold-stress effects and control of energy, protein, fat, and nucleic-acid metabolism in aquatic animals, especially in fish, with the ultimate goal of reducing or preventing cold-stress related damage. Future studies should investigate these questions in a multidisciplinary fashion, incorporating molecular techniques to understand cold-stress effects on the molecular, cellular, and tissue levels. Multiple measures are necessary to develop sustainable, lower-risk fisheries, including the improvement of environmental conditions, genetic engineering and breeding cold-resistant species, along with increasing cold-stress resistance through dietary (supplementation of proteins, polyunsaturated fatty acids, trace elements, and other nutrients).