Abstract:

Abstract:

Abstract:Keystone species play an important role in structural stability of a community and changes in diversity. Using Yellow Sea autumn survey data for 1985, 2001, 2009, and 2018, we constructed the topological structure of the food-web network of the Yellow Sea fish community and analyzed the interdecadal changes of keystone species in the Yellow Sea fish community. The food webs included 67~103 different fish species and 300~449 prey-predator relationships. The structural density of these food webs ranged from 0.198 to 0.227, and interspecies connectivity ranged between 0.044 and 0.074, consistent with fish communities under natural conditions. The keystone species of the autumn fish community in the Yellow Sea from 1985 to 2018 were Engraulis japonicus, Lophius litulon, and Larimichthys polyactis, remaining unchanged for nearly 30 years. E. japonicus is the keystone prey in the fish community, while L. polyactis as a species that controls the fragmentation of community, both resources decline. As the keystone predator, L. litulon resources have gradually increased. From 1985 to 2018, the dominant species in the Yellow Sea changed significantly in autumn, gradually shifting from L. polyactis and Pampus argenteus to Harpadon nehereus, Liparis tanakae, and E. japonicus. The Margalef richness index (Rw, Rn) and Shannon diversity index (H'w, H'n) calculated by weight and quantity have gradually decreased and have rebounded significantly in 2018, while Pielou evenness index (J'w, J'n) fluctuated slightly. The keystone species in the Yellow Sea did not change in autumn; however, the dominant species changed significantly, while the community structure fluctuated slightly but still remained in a relatively stable state.

Abstract:Based on field data of nutrients observed in Laizhou Bay during May and August 2018, using the methods of nutrient restriction and potential eutrophication assessment standards, the distribution characteristics of nitrogen, phosphorus, and silicate in the Laizhou Bay were analyzed and discussed. The results showed that the concentrations of DIN ranged from 1.64 μmol/L to 106.36 μmol/L with an average content of 24.18 μmol/L and were significantly higher in May. The concentrations of PO34–-P ranged from “not detected” to 2.010 μmol/L with an average content of 0.182 μmol/L, and were significantly higher in August. The concentrations of SiO23–-Si ranged from 0.97 μmol/L to 78.93 μmol/L with an average content of 18.30 μmol/L and were also significantly higher in August. The areas with higher values for DIN, PO34–-P, and N/P ratio were mainly located within the sea area near the Xiaoqing River and Yellow River estuaries in the west of Laizhou Bay. The areas with higher values for SiO23–-Si、Si/N ratio, and Si/P ratio were mainly located at the bottom of Laizhou Bay and near the coast of Longkou-Laizhou. The nutrient structure analysis indicated that availability of PO34–-P was limited in May and August while that of SiO23–-Si was limited in May. The spatial distribution of nutrients in the Laizhou Bay was mainly influenced by the terrestrial inputs, especially during periods of high water levels. This observation aligns with the higher nutrient content of the Xiaoqing River and Yellow River estuaries. SiO23–-Si and PO34–-P were the main limiting factors of primary productivity in May, while DIN and PO34–-P were the main limiting factors of primary productivity in August. The horizontal distribution of nutrient structure restriction indicated that it was easy to cause red tide in Laizhou-Zhaoyuan culture area in spring and Dongying-Weifang culture area in summer. The limited primary productivity in the Laizhou-Zhaoyuan area during summer could substantially influence the aquaculture in this area.

Abstract:The genetic diversity of Jinhu grouper hybrids (Epinephelus fuscoguttatus ♀ ×E. tukula ♂) and their parents were analyzed via microsatellites. The results detected 215 alleles at 20 microsatellite loci, with an average polymorphic information content (PIC) of 0.638 6. The highest average effective allele number (Ne) was measured in E. fuscoguttatus (4.367 9), followed by the hybrids (3.370 6) and E. tukula (2.412 8). The average observed heterozygosity values (Ho) were 0.385 5 (E. tukula), 0.474 9 (E. fuscoguttatus), and 0.473 6 (hybrids), and the average PIC values were 0.491 9, 0.645 3, and 0.555 1, respectively. The three grouper populations were averaged to calculate the inbreeding coefficient (Fis; 0.228 0), total population inbreeding coefficient (Fit; 0.344 1), genetic differentiation coefficient (Fst; 0.150 4), and gene flow (Nm; 1.411 9), but differentiation among the populations was also observed. Cluster analysis, performed by the unweighted pair group method with arithmetic mean, placed the hybrids and E. fuscoguttatus into one group, indicating a close relationship. Prior studies showed that the Jinhu grouper is genetically diverse, which allowed for selective breeding, hybrid generation, and exploration of the genetic basis of heterosis.

Abstract:This study explored the roles of ATPase Na+/K+ transporting subunit alpha 3 (ATP1A3) and ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2B1) in the low-salinity stress response of Trachidermus fasciatus (roughskin sculpin). ATP1A3 and ATP2B1 gene sequences were obtained from the T. fasciatus transcriptome data, and a phylogenetic analysis was performed. Roughskin sculpins were subjected to two acute osmotic salt treatments (salinity change rates of 27/h and 1.1/h) to induce stress, and the expression patterns of the target genes in four tissues (gill, intestine, kidney, and liver) were analyzed by quantitative real-time PCR (qRT-PCR). The phylogenetic analysis showed that the ATP1A3 and ATP2B1 genes formed an independent cluster, and the T. fasciatus ATP1A3 and ATP2B1 proteins shared a high identity with those of Perciformes and Pleuronectiformes species. The teleost ATP1A3 and ATP2B1 proteins formed a single distinct lineage from other vertebrates. For both genes, each tissue had its own gene expression pattern in response to salinity. In the gills, ATP1A3 expression increased and then decreased under chronic salinity stress, but ATP1A3 expression significantly decreased under acute stress. ATP2B1 expression increased under chronic and acute stress (with a significant increase after 24 h of chronic stress). In the intestines, ATP1A3 expression significantly decreased after 24 h in both treatments, but the ATP2B1 expression significantly increased under chronic stress and increased after 24 h of acute stress. In the kidneys, the expression levels of both genes peaked after 24 h of chronic stress and increased significantly under acute stress. ATP1A3 and ATP2B1 expression was undetected in the liver under chronic stress, but under acute stress, incremental expression was detected for both genes. These results demonstrate that the ATP1A3 and ATP2B1 gene expression profiles are affected by salinity stress but not in the same way. These findings provide a foundation for future investigations into the role of ATP1A3 and ATP2B1 in fish osmotic pressure regulation and the molecular mechanisms underlying migratory fish salinity adaptation.

Abstract:Melanin commonly exists in animal tissues. The animal body surface has different colors and abundant pigment patterns due to differences in the distribution and development of melanin and related cells. Different pigment patterns have important functions such as biological camouflage, courtship, etc., especially in fish. Cynoglossus semilaevis, one of the dominant aquaculture flatfish species in China, often suffers from two body color anomalies, i.e. melanism and albinism, which have hindered the development of high-quality seeds and aquaculture. In this study, cDNA sequences of two color-related genes (i.e. TYR and DCT) were cloned and phylogenetically analyzed. Further, expression levels of both genes were analyzed in different stages and in different tissues. The cDNA sequence length of TYR gene coding region is 1620 bp, encoding 539 amino acids. The length of the cDNA sequence in the coding region of DCT gene is 1551 bp, encoding 516 amino acids. In this study, it was found that the expression levels of TYR and DCT were high in fry that were less than 20-day-old, especially during the critical period of metamorphosis (15 to 20-day-old), and decreased to a very low level at 30-day-old. In other skin tissues, the expression levels of these two genes were highest in normal skin on the ocular side and in dark skin on the blind side, and extremely low in albino skin on the ocular side and in normal skin on the blind side. Among other tissues at other times, the highest expression was in the eye, followed by the liver, with very low expression in the spleen and muscles. Results show that TYR and DCT genes are the key genes for blind-side melanogenesis and ocular-side color maintenance in C. semilaevis. This study provides an important basis and reference for identifying the mechanism of color anomaly in C. semilaevis.

Abstract:This study aimed to investigate the methods of isolation, culture, and identification of melanophores and xanthophores in polychromatic midas cichlids (Amphilophus citrinellus) and to thus observe the growth characteristics of pigment cells in fish. In this study, the caudal fin, scales, and skin tissues of A. citrinellus were used as the experimental material. To separate the pigment cells, the tissues were digested in trypsin solution and collagenase solution for 6 h and 12 h, respectively, at 25℃~28℃. After the cell suspension was filtered through a stoma nylon net, xanthophores and melanocytes were separated and collected by means of 35%-45%-55% Percoll multilayer density gradient centrifugation. K-SFM medium was used for primary cell culture and cell purification to inhibit the growth of fibroblasts and keratinocytes. DMEM supplemented with phorbol ester (TPA), double resistance, and bEGF, etc., was used for the subculture of pigment cells. The morphology of the cells was observed by L-DOPA staining and transmission electron microscopy, and the cells were identified by molecular markers. The results showed that when the cells were separated, the melanocytes were located at the boundary of the 45% and 35% Percoll reagent layers, while xanthophores were located on top of the 35% Percoll layer. Both pigment cells condensed into flocculation. Transmission electron microscopy showed that the melanocytes contained a large number of melanosomes, while the yellow cells contained the MTX and carotenoid vesicles. The melanocytes were positive under L-DOPA staining. The 10th generation of pigment cells was used for PCR detection of the body color related genes MC1R, TYR, and EDNRB, which showed strong specificity of gene amplification bands, indicating that the two pigment cells obtained had good biological activity. In this study, methods for the isolation, culture, and identification of melanophores and xanthophores of polychromatic midas cichlids were successfully established, providing a cell model for further research on the molecular mechanisms of body color cell differentiation and body color formation in fish.

Abstract:Fish cell culture is an important model for germplasm preservation, gene function analysis, and cell engineering breeding. Therefore, it is of great significance to establish cell lines for the endangered Schizothorax eurystomus. The aim of this study was to establish a kidney tissue cell line for S. eurystomus, for which mesonephric tissue was used. The primary cell culture was initiated by tissue block transplantation and the subculture was carried out after the cells were stabilized. A kidney cell line (EUM10) was established. To analyze the cryopreservation and resuscitation ability of renal cell lines and the optimal growth conditions for S. eurystomus, PCR was used to detect contamination and mitochondrial gene fragment analysis was used to identify the source of the cell. The results showed that the optimal medium for S. eurystomus kidney tissue cells was DME/F-12, the optimal temperature was 25℃, and the optimal serum concentration was 20%. The cells grew in suspension and the growth curve was S-shaped. The growth curve shows that the population doubling time of the kidney tissue cell line cells in the 10th generation was 23.26 h. After 6 months of cryopreservation with liquid nitrogen, the recovery/survival rate was 91.91% (6th generation). S. eurystomus shares 99.81% of the gene sequence identity with a split-bellied fish from Tarim, which proves that the cell line originates from a wide-mouthed split-bellied fish. In this study, a kidney cell line for S. eurystomus was successfully established. Kidney cell lines can be used as in vitro systems to study various aspects of fish, such as cytogenetics and gene function analysis. This study provides the basic data for the initial research into and breeding of S.eurystomus.

Abstract:Discogobio yunnanensis is a small Cyprinid fish, well adapted to flowing water and mainly distributed in Jinsha River, Yuanjiang River, Nanpanjiang River, and Yangtze River. Due to the long-term adaptation to the lotic environment, the lips and related structures have changed accordingly. The lower lip gradually stretches backwards, and the chin uplift is specialized into a mouth-sucker structure with horseshoe folds. However, D. yunnanensis is also named "stone fish" because it likes to scrape the surrounding clumps attached to the stone surface. At water temperature of (20.0±0.5)℃, the unique characteristics in the morphology and histology of the sucking disc of D. yunnanensis larvae and juveniles were observed using light and scanning electron microscopy to understand the characteristics of the sucking disc. The evolution of the sucking disc was classified into three development stages: the pre-forming stage, the forming stage, and the perfecting stage. The primordium of the sucking disc was not formed in the pre-forming stage. In the forming stage, the sucking disc primordium appeared; the lower lip flap, granulation, lateral projection, and the main body of sucking disc were connected as a whole with few gaps, and the prototype of the sucking disc was formed. In the perfecting stage, the gaps disappeared, and the sucking disc was fully developed. There were no mastoids at either side of horseshoe swollen and the leading edge of central pad; however, the other areas of the sucking disc were covered with mastoids of different sizes. Taste buds were mainly distributed in the rostral fold, maxillary barbel, rostral barbel, protuberance of lips, central pad, and corner of the mouth.

Abstract:Light is one of the main factors affecting fish activity, feeding, and growth, and has thus been an active focus of marine research. The aim of this study was to investigate the selectivity of light spectra and intensity in Schizothorax wangchiachii using wild and cultured fishes. The study investigated the effects of light on the growth performance and physiological characteristics of S. wangchiachii. S. wangchiachii were cultured in a 12-replicated recirculating aquaculture system with four different lighting conditions (natural light, blue light, green light, and yellow light) for 8 weeks. The results showed that S. wangchiachii were attracted to the yellow, red, and green light, with blue light having a negative effect on species. No significant differences were found (P<0.05) between the phototaxis behaviors of S. wangchiachii in response to different light intensities (22.6~64.7 lx) under green and yellow light. The results showed that light colors have a notable impact on the growth rate and survival rate of S. wangchiachii (P<0.05). The yellow light showed better trapping ability but did not improve the growth and survival rate of S. wangchiachii, however, the survival rate of S. wangchiachii was only 46.7% in the blue light treatment group. In addition, there was no significant difference between cortisol levels of the natural light and yellow light treatment groups, but they were significantly lower than those of the blue light treatment group. In conclusion, better growth performance was obtained with yellow and natural light treatments, and the survival rate of S. wangchiachii was more than 85%, both faring significantly better than the blue light treatment. Blue light increased the stress levels of S. wangchiachii and led to a decreased survival rate. These results can provide scientific basis for the wild harvest of fish, solutions for fish passage around hydraulic engineering construction, and in the selective breeding of S. wangchiachii.

Abstract:This study investigated changes in fatty acid and amino acid contents in the muscle and hepatopancreas of red crayfish (Cherax quadricarinatus) at different growth stages, i.e., before the second sex development (90 days), at maturation of second sex (140 days), and at gonadal maturity (180 days). Sixteen types of amino acids in the muscle were found, and the content of essential amino acids (EAAs) at 90 days was significantly higher than the other two periods (P<0.05). However, the content of EAAs and non-essential amino acids in the hepatopancreas of crayfish decreased gradually. Among fatty acids, the saturated fatty acids (SFA) in the muscle showed a decreasing trend; the monounsaturated fatty acid (MUFA) content first increased and then decreased, reaching peak content of (25.69±0.42%)% at 140 days, whereas the polyunsaturated fatty acid (PUFA) content increased from 90 to 180 days. Furthermore, the SFA content in the hepatopancreas showed no significant difference among the three growth periods (P>0.05); the MUFA content first increased and then decreased, peaking (37.44±0.59)% at 140 days, while the PUFA content showed the opposite trend. Moreover, the evaluation of nutritional value revealed that the EAA index (49.96%) of the muscle of 180-day-old (marketable) red crayfish was higher than the FAO/WHO score model (31.50%) and the whole egg protein model (43.10%), which is considered an ideal source of high-quality protein. In conclusion, the consumption of amino acids and fatty acids in the muscle and hepatopancreas is different. These results provide a theoretical reference for understanding the nutritional requirements of red crawfish at different stages of growth and development.

Abstract:Phenanthrene (PHE) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems. It has strong hydrophobicity and is often adsorbed on sediment and suspended particles in water, which has potential toxicity to aquatic organisms. Mussels (Mytilus coruscus) are widely distributed in the ocean and live in a fixed environment. They are filter feeders and have a strong accumulation of organic pollutants in water. They are often used as indicator organisms in monitoring marine environmental pollution. In this study, enrichment (10 d) and release (5 d) tests of PHE with different exposure concentrations were carried out. Three groups of PHE (4 μg/L, 20 μg/L, and 100 μg/L) and artificial seawater group (control group: 0.01% acetone) were set up. Three parallel experimental replicates were set up in each group, and 16 mussels were placed in each group. Two mussels were randomly selected on the 1st, 3rd, 6th, 10th, 12th, and 15th day. Their visceral mass, outer membrane, and closed shell muscle tissue were separated and stored at –80℃ for identification and analysis. Enrichment and release of PHE and the change in HSP70 gene expression in visceral mass, outer membrane, and closed shell muscle tissue were analyzed using HPLC and qPCR, respectively. Results showed that the concentration of PHE in the three tissues of mussels was in this order: visceral mass > outer membrane > closed shell muscle at the same time and concentration. The enrichment content of PHE in the three tissues increased with an increase in time and concentration, owing to the higher n-octanol/water partition coefficient and fat solubility of PHE, filter feeding life of mussels, and the fact that tissues with high fat content are more likely to enrich PHE. For the release test, the PHE content in the three tissues of mussels decreased rapidly in the early stage of release; however, on the 15th day, the residual amount of PHE in the three tissues was still higher than that in the control group. This is because the release of PHE in different tissues was mainly controlled by diffusion driven by thermodynamics, metabolic activity regulated by enzyme system, and excretion when mussels were in the state of water release. In addition, PHE content in mussel tissues began to decrease rapidly in the early stage of release and gradually decreased with the extension of release time. Furthermore, HSP70 was induced to enhance the anti-stimulation and survival ability of the organism under PHE stress; the expression of HSP70 mRNA in mussels was tissue-specific, and the expression level of HSP70 in the outer membrane was the highest. These results provide a reference for the study of enrichment kinetics and toxic mechanisms of PHE in shellfish.

Abstract:To study the structure and function of MHC (major histocompatibility complex) in Pelodiscus sinensis, we obtained the full-length cDNA of MHCⅡα using RACE-PCR (rapid amplification of cDNA ends PCR) technology. The resulting sequence was 1296 bp in length, including an ORF (open reading frame) region of 807 bp. The peptide-encoded MHCⅡα gene could be divided into four parts, including the signal peptide, MHCⅡ alpha domain, IGc1 domain, and transmembrane region. A neighbor-joining tree showed that the MHCⅡα gene from P. sinensis and Chrysemys picta bellii cluster into one cluster. The P. sinensis gene had a close genetic-relationship with C. picta bellii and a farther genetic-relationship with mammals, aves, and fish. Using qRT-PCR in all 8 tissues, the highest expression of the MHCⅡα gene was observed in the spleen, heart, liver, and intestine, while the lowest level was found in muscle. Meanwhile, MHCⅡα mRNA expression levels were significantly up-regulated in the liver and intestine (after 12 h), the spleen (on the 1st day), and the kidney (on 1st and 5th day) after being infected with Aeromonas hydrophila. The liver, spleen, intestine, and kidney are closely related to immunity, which indicates that this gene has important effects on the immune response.

Abstract:Similar to hemocytes in other species, coelomocytes of sea urchins play an important role in the immune system. A verification of the recovery pattern and origin of coelomocytes would be vital in understanding its immune mechanism. Axial organ is a glandular organ that has been thought to be the hemopoietic tissue of sea urchin. Using monoclonal antibodies against coelomocytes, we demonstrated in a previous study that axial organ could serve as a storage tissue of coelomocytes. We further investigated the recovery pattern of coelomocytes in the sea urchin Strongylocentrotus intermedius and the changes in the axial organ during the recovery phase. Coelomocyte density was examined at 6 h, 12 h, 18 h, and 24 h after manual extraction of 10% body weight of celomic fluid. Histology of the axial organ was observed, and cell proliferation activity was detected with a monoclonal antibody against Ki-67 (cell proliferation antigen). Results showed that the average coelomocyte density at 6 h was significantly lower than that before extraction; it gradually decreased at 12 h, with a density slightly lower than the initial level, with a recovery value of (92.78±29.40)%. Average coelomocyte was significantly higher than the initial level at 18 h, with a recovery value of (137.08±32.40)%, and was significantly higher than that at 6 h and 12 h (P<0.05). Subsequently, the coelomocyte density gradually decreased. The inter-tissue of the axial organ declined and cavitation occurred after coelomocytes were lost. The outer epithelial layer became loose and broke off, and vacuolation was observed in the bulges of the epithelial layer. The inter-tissue increased at 18 h, and newborn tissue appeared clearly at 24 h. Using the Ki-67 antibody, we found a cell production signal in the axial organ from the normal sea urchin and the signal was significantly enhanced after 18 h. Our results indicated that the restoring mechanism of coelomocytes could initiate rapidly after coelomocyte loss, while histological changes and cell production activity enhancement may occur in the axial organ. The present study provides valuable references for further research on the origin and ontogenesis of coelomocytes.

Abstract:Low-temperature-adapted alginate lyase has unique advantages for alginate oligosaccharide preparation and brown algae processing. Producing a low-temperature-adapted alginate lyase with better stability remains an urgent need for industrial applications. In this study, to screen for a low-temperature- adapted alginate lyase with good stability, we identified and isolated marine bacteria, by cloning and analyzing enzyme-encoding gene and secretory expression and purification of enzyme. The effects of different factors on enzyme activity and stability, as well as analysis of enzymatic hydrolysis products were evaluated. Results showed that the marine bacterium SJ-H-12 is capable of secreting low- temperature-adapted alginate lyase when cultivated with alginate as the sole carbon source. An evolutionary tree was constructed based on 16S rDNA sequences, and the strain was identified as Yangia sp. SJ-H-12. Furthermore, the gene encoding the enzyme ALYYA was identified as belonging to the PL5 alginate lyase family. The gene was secreted and expressed in Yarrowia lipolytica, a food-grade host. The activity of recombinant ALYYA reached 34.2 U/mL; the molecular weight of ALYYA was about 39.0 kDa, and it had a strong PolyM preference. ALYYA showed more than 80% vitality at 25℃~35℃, with peak vitality at 30℃; it demonstrated good stability in the pH range of 5.0~10.0, with more than 60% vitality remaining after incubation. NaCl could activate the activity of ALYYA at a concentration of 0~2.0 mol/L. The products of ALYYA-degraded alginate are mainly disaccharides and small amounts of monosaccharides and trisaccharides, resulting from its endoalginate lyase activity. In summary, a low-temperature-adapted alginate lyase-producing bacterium was screened, and most of the alginate lyases found were mesophilic enzymes. This study showed that ALYYA is a typical low-temperature- adapted alginate lyase with excellent enzyme activity and stability, providing reference data for the screening and industrial development and utilization of low-temperature-adapted alginate lyase.

Abstract:The type Ⅵ secretion system (T6SS), a protein secretion system generally found in gram-negative bacteria, plays an essential role in virulence, interspecific competition, and environmental adaptability. Hemolysin-coregulated protein (Hcp), an extracellular component of T6SS, is released into the medium and therefore may serve as a marker of a functional T6SS apparatus. RpoS, the σ subunit of RNA polymerase, is involved in the regulation of bacterial growth and stress response. To explore the regulatory effect of rpoS on Vibrio anguillarum T6SS, an MHK3∆rpoS mutant strain was constructed; phenotypic changes were detected using the lacZ reporter gene to construct fusion strains and the changes in hcp1 and hcp2 expression at the transcriptional level were detected using the ONPG (o-nitrophenyl-ß-D-galactopyranoside) method. Western blotting was performed to quantitatively analyze the changes in the mutant Hcp expression at the translational level. The change in the bactericidal activity of the mutant strain was detected via a bacterial antagonistic experiment. The results showed that the growth, mobility, gelatinase activity, and caseinase activity of the MHK3∆rpoS mutant strain showed no significant difference compared with the wild-type strain MHK3 (P>0.05). The biofilm-forming ability significantly increased at the prophase after mutation in rpoS (P<0.05). The transcriptional levels of hcp1 and hcp2 in each growth phase were significantly higher in MHK3∆rpoS than in MHK3 (P<0.01), with highest increases of 1.79-fold and 1.94-fold compared with MHK3, respectively. At the translational level, the secretion of both intracellular and extracellular Hcp in MHK3∆rpoS significantly increased (P<0.05), with highest increases of 1.59-fold and 1.31-fold, respectively. Meanwhile, the bactericidal activity of MHK3∆rpoS against Escherichia coli E5 was about 1% of MHK3. Studies have shown that rpoS has no significant regulatory effect on some phenotypes of MHK3; however, it negatively regulates the biofilm-forming ability at the prophase. This is different from the results of the research on V. anguillarum M3 that posits that rpoS-mediated regulation of the same phenotype in different strains varies. Furthermore, at the levels of transcription and translation, rpoS negatively regulates the expression of Hcp; however, it shows a positive regulatory effect on the bactericidal activity of MHK3. This indicates that the strength of the bactericidal activity of the strain is not directly related to the expression and secretion of Hcp. This study provides innovative ideas and enriches the theoretical basis for further elucidating the regulatory mechanism of T6SS and T6SS-mediated bactericidal activity.

Abstract:Water bodies with nutritional differences have different plankton composition, which may lead to different food composition for bighead carp, thus affecting the meat quality. At present, the annual output of bighead carp in China is approximately 3.1 million tons, and its feeding amount occupies a dominant position in fish farming in China. As the economy develops, consumers demand better food quality and especially high nutritional value. Therefore, this study explored the changes in the nutrient composition and content of bighead carp in different nutritional types of water. Using a single-factor experiment design, we selected bighead carp that were raised in high-, medium-, and low-nutrition natural reservoirs and artificial ponds (body length: 44.98~48.98 cm; weight: 1946.37~2289.32 g) and determined the physical properties and nutritional composition of the meat. The results showed that different nutritional types of water had no significant effect on the pH, a* value, shear force, water content, and ash content (P>0.05). Compared with artificial ponds, the L* value and cooking loss rate in the natural reservoir and the crude fat content in low-nutrient reservoir were significantly reduced (P<0.05), whereas the crude protein content in medium- and low-nutrient reservoirs were significantly decreased (P<0.05). When comparing reservoirs of different nutrition types, the crude fat content in high- and medium- nutrient reservoirs were significantly greater than low-nutrient reservoirs (P<0.05). Additionally, the crude protein content in high-nutrient reservoirs was significantly higher than medium- and low-nutrient reservoirs (P<0.05). Seventeen amino acids and 13 fatty acids were detected in bighead carp reared in natural reservoirs and artificial ponds. However, levels of essential amino acids, palmitic acid, myristic acid, and eicosaenoic acid in the natural reservoir were significantly higher than in the artificial pond (P<0.05). Taken together, the meat of bighead carp raised in natural reservoirs has the advantages of high quality meat color, strong water-holding capacity, low fat, and high essential amino acid content.

Abstract:The sea cucumber Acaudina molpadioides is a low-value aquatic product of the ocean. To increase its value, in this study, dried body walls of low-value A. molpadioides were selected as research material, from which the polypeptides were extracted by employing twin-screw extrusion with sodium sulfite-assisted treatment. The extraction rate of polypeptides and free sulfhydryl groups, and protein solubility from extrudate of A. molpadioides were determined. Polypeptides were separated from the extrudate and purified with DA201-C type microporous adsorption resin, and the molecular weight distribution and antioxidant ability of the purified components were determined. The results showed that for the A. molpadioides treated with twin-screw extrusion-assisted 3.0% sodium sulfite, the polypeptide extraction rate, free sulfhydryl content, and protein solubility were (57.12±0.62)%, (110.32±0.07)%, and (28.72±0.13)%, respectively, which were significantly higher (P<0.05) by (7.66±0.35)%, (105.32±0.01)% and (4.91±0.15)%, respectively, compared with those of the control check (CK). Moreover, the molecular weights of the purified polypeptides component I and Ⅱ were 1000~3000 u and <1000 u, respectively. Additionally, comparing the A. molpadioides polypeptides with those in the CK group, twin-screw extrusion-assisted 3.0% sodium sulfite extraction group, purified component I, and purified component Ⅱ, the IC50 values of DPPH radical scavenging rate were 25.51 mg/mL, 12.72 mg/mL, 6.58 mg/mL, and 9.02 mg/mL, respectively, and the IC50 values of the superoxide anion radicals scavenging rate were 25.56 mg/mL, 13.51 mg/mL, 11.87 mg/mL and 8.44 mg/mL, respectively. The results of oxidation damage protection test suggested that for purified component I, the optimal concentration for the survival rate of LO2 cells was 80 μg/mL, with (20.33±0.41)% increase compared with that in the injured group; as for the purified component Ⅱ, it was 20 μg/mL with (17.07±1.18)% increase than that in the injured group. In summary, the twin-screw extrusion-assisted sodium sulfite treatment on A. molpadioides improved the extraction rate of polypeptide and the extracted polypeptide possessed enhanced antioxidant activity.

Abstract:Bacillus subtilis is used as a probiotic bacterium in aquaculture and has been explored as a live carrier for the expression and oral delivery of antigen proteins. Flavobacterium columnare, a gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in fish. It is ubiquitous in aquatic environments and causes disease in a variety of freshwater fish worldwide, which leads to severe economic loss. In a previous study, we discovered a protective antigen gene, lip, against F. columnare. In this study, the lip gene was inserted into a shuttle expression vector pBE2R, and then expressed as a recombinant fusion protein in B. subtilis WB800. The expressed protein of 32-kDa, which was estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blotting, was consistent with the molecular weight deduced from amino acid sequence. Grass carp (Ctenopharyngodon idella) was immunized through oral administration of B. subtilis WB800 (pBE2R-lip). The effects of protection and immune responses were evaluated. Enzyme-linked immunosorbent assay results showed that lip-specific antibodies could be detected in the sera of immunized fish, and the antibody levels were highest in the fourth week, with an average of 1:117. The outcome of immunization with B. subtilis WB800 (pBE2R-lip) resulted in a relative percent survival of 52.4% against F. columnare. After being immunized via oral administration, the quantity of B. subtilis in the hindgut of grass carp was measured using spread plate count method. Results showed that B. subtilis could not colonize the gut of grass carp. Our study demonstrated that oral administration of B. subtilis expressing lip gene could produce an effective immune response and offer good resistance against F. columnare.

Abstract:Cyprinid herpesvirus 2 (CyHV-2) and Cyprinid herpesvirus 3 (CyHV-3) mainly infect crucian carp (goldfish), common carp, koi, and their hybrids. They are highly infectious and pathogenic viruses and can seriously damage the culture of Cyprinid fishes. In order to establish a rapid, and efficient method for simultaneously detecting the two viruses, three pairs of specific primers were designed based on the conserved sequences of the genes encoding DNA polymerase (CyHV-2, 3), helicase (CyHV-2), and thymidine kinase (CyHV-3). By optimizing the reaction conditions and system for the triplex PCR, a detection method for identifying different types of carp herpesviruses was established and validated using the Carassius auratus and Cyprinus carpio tissue samples preserved in the laboratory. The results showed that the triplex PCR method amplified only three specific bands from the herpes virus positive carp samples, showing good specificity. Using the cloned plasmid as a template with 10-fold dilution, the detection limit of the method was 2.85×102 copies/μL, showing high sensitivity. A total of 122 samples of crucian carp and 60 samples of common carp were analyzed by this method and the detection results were consistent with those of the national standard (GB/T 36194-2018) and industry standard (SC/T 7212.1-2011). In conclusion, the newly established triplex PCR method is not only highly accurate and sensitive, but can also detect two types of carp herpesvirus simultaneously, thereby effectively improve the detection efficiency.

Abstract:In order to analyze the quality status and existing problems (loss of juice, discoloration of meat, change in taste, etc) of frozen scallops sold in Shanghai, we explored the quality evaluation methods of frozen scallops in the market and the key factors that lead to their quality deterioration during frozen storage. In this study, 11 kinds of frozen scallop products sold in Shanghai supermarkets were sampled based on 5 common influencing factors such as freezer temperature, frozen storage time, product form, packaging method, and placement position. We measured 10 quality indicators such as whiteness, thawing loss, cooking loss, water holding capacity, resilience, cohesiveness, springiness, chewiness, hardness, and sensory score. Factor analysis was used to construct a quality evaluation model for frozen scallops and was combined with multiple linear regression analysis to determine the key factors affecting the quality of frozen scallops. Results show that there is a certain correlation between the 10 quality indicators of frozen scallop products. Three factor components were extracted through factor analysis, and the cumulative variance contribution rate was 73.33%. It can replace the original indicators to comprehensively evaluate the quality of frozen scallop products. Quality evaluation model of the frozen scallop was established by factor analysis: f=0.467f1+0.302f2+0.231f3. The common factors of f1 were springiness, resilience, cohesiveness, cooking loss, and water holding capacity; common factors of f2 were hardness, whiteness, and chewiness; and common factors of f3 were thawing loss and sensory score. Furthermore, multiple linear regression analysis showed that frozen storage time, freezer temperature, and product form are the key factors that affect the quality of frozen scallop products; the other two factors have no significant influence. These results suggest that methods based on factor analysis and multiple linear regression analysis can be used to evaluate the quality of frozen scallops and analyze the factors influencing quality deterioration.

Abstract:The 86 spotted sea bass (Lateolabrax maculatus) used as experimental subjects were collected from eight main cultured populations, namely Shandong Province (Qingdao and Dongying), Zhejiang Province (Ningbo), Fujian Province (Fuding, Meiling Town of Zhangzhou, Qiaodong Town of Zhangzhou, and Dongshan Town of Zhangzhou), and Guangdong (Doumen District of Zhuhai). Based on the reported genetic background of wild populations of spotted sea bass, we conducted a genetic structure analysis on the eight cultured populations using the double-digest restriction-site-associated DNA tag sequencing and simplified genome sequencing analysis technology. The results of a principal component analysis and genetic ancestry analysis (the best clustering K value is 2) showed that there was little genetic divergence between the eight cultured populations and the wild populations of Tianjin, Yantai, and Wendeng in Shandong Province, indicating that the germplasm sources for cultured spotted sea bass are mainly the Yellow Sea and Bohai Sea. The results of the genetic differentiation analysis among the eight cultured populations showed that there was significant genetic divergence between the Meiling Town and Qiaodong Town populations in Zhangzhou and other cultured populations, indicating that a certain degree of differentiation appeared within the identified cultured populations. The results provide a reference for the analysis of the genetic structure of cultured spotted sea bass populations from Shandong to Guangdong (north to south), and provide a basis for the protection and improvement of spotted sea bass germplasm resources and breeding of spotted sea bass.

Abstract:The gonadogenesis, the development and differentiation of larvae and juveniles from 1~214 days post hatching (dph) and 18-month-old female and male spotted sea bass (Lateolabrax maculatus) were studied. The relationship between the expression of sex-related genes (cyp11b and cyp19a1a) and sex in the process of gonadal differentiation was discussed. The results showed that at 30 dph [(1.28±0.10) cm], primordial germ cells (PGCs) were first observed around the peritoneal membrane at the front end of the mesorenic duct, indicating that the time before 30 dph was the critical period for PGCs to migrate to the genital ridge. At 55 dph [(2.45±0.19) cm], a pair of symmetrically distributed primitive gonads was observed, indicating that the primitive gonads of the juvenile spotted sea bass were formed between 30 and 55 dph [(1.28~2.45) cm]. Between 55~180 dph [(2.45~12.28) cm], the primordial gonads continued to grow and remained in an undifferentiated state. After 180 dph, the gonads began to differentiate; at 195 dph [(14.54±1.54) cm], we observed that the testis began to differentiate, while the ovary began to differentiate at 205 dph [(15.86±0.94) cm]. The anatomical differentiation of the gonads was earlier than the cytological differentiation in spotted sea bass. The gonad of 18-month-old spotted sea bass developed to stageⅡ. The expression level of the sex differentiation-related gene cyp19a1a in the ovary of spotted sea bass was higher than that of the contemporaneous testis, indicating that it played a key role in the differentiation and maintenance of the ovary. The expression level of cyp11b at stageⅡ testis of 18-month-old juveniles was significantly higher than in the contemporaneous ovary and stage Ⅰ testes, suggesting that it played a crucial part in the differentiation and maintenance of testes. The results not only enriched our understanding of the reproductive physiology of spotted sea bass, but also provided a scientific basis for the study of sex selection technology.

Abstract:Yellowtail amberjack, Seriola quinqueradiata, is a globally distributed, economically important, marine pelagic fish species belonging to the same genus as S. aureovittata and S. dumerili. It is a popular table fish worldwide because of its flavorsome flesh and high nutritional value. Recently, we made a breakthrough in triggering the natural spawning of S. quinqueradiata by manipulating photothermal regimes in China, and produced over 7700 juveniles with an average total length (TL) of 147 mm in an indoor tank culture system. Meanwhile, the morphological and quantitative characteristics and the growth and development of S. quinqueradiata during the early life history stages were observed and described for the first time. The mature eggs of S. quinqueradiata are transparent, spherical, and buoyant, with a single oil globe with a diameter of approximately 1.26~1.36 mm. The embryos hatched out at approximately 37 h 40 min post hatching at a water temperature of (22.0±0.5)℃. The embryonic development process can be divided into eight stages: pre-cleavage, cleavage, blastula, gastrula, neurula, organogenesis, muscle effect, and hatching. The morphology of embryonic development has been described previously. The TL of the newly hatched larvae was (4.03±0.27) mm with an oval yolk sac, which accounted for 3/8 of the TL. The TL of larvae 3 days after hatching (DAH) was (16.23±1.61) mm, and the mouth opened and larvae entered the mixed nutrition period. The first food was a rotifer. The TL of larvae at 6 DAH was (4.93±0.17) mm; here, the yolk sac was exhausted and the swim bladder started to inflate. The TL of larvae at 10 DAH was (5.21±0.23) mm, and the larvae entered the exogenous nutrition stage. The TL of larvae at 15 DAH was (6.24±0.66) mm, and the end of the spine began to bend. The TL of larvae at 25 DAH was (10.25±1.35) mm, and the spine bending process was completed; thereafter, the larvae began to feed on Artemia nauplii. The TL of juveniles at 30 DAH was (16.23±1.61) mm, which is when commercial feed conversion started, and the juveniles began to feed well on the commercial diet at 40 DAH when the TL reached (28.07±2.32) mm. The TL of juveniles at 65 DAH was (81.49±5.11) mm, which was when the juvenile morphology was similar to the adults. Furthermore, the suitable temperature for embryonic development was determined to be 22℃~24℃. The results provide technical support regarding artificial breeding and seedling production technology of S. quinqueradiata that could boost the development of the S. quinqueradiata farming industry in China.

Abstract:Yellowtail kingfish Seriola aureovittata is a globally distributed, circumtropical pelagic fish of importance in both commercial fisheries and aquaculture. It swims fast and has fierce behavior under artificial culture conditions. To develop a suitable manipulation technique with low stress for yellow tail kingfish, the anesthetizing effects of two anesthetics, MS-222 and clove oil, on one-year-old yellowtail kingfish under different temperature conditions (20℃ and 24℃) were studied. The optimal anesthesia times, recovery times, and anesthesia dosages of the two anesthetics were determined based on the discrimination of swimming behavior and investigation of serum hormones levels, including cortisol, adrenaline, and glucose. According to the behavioral characteristic changes of the yellowtail kingfish during anesthesia and recovery, the anesthesia process was divided into seven stages, whereas the recovery process was divided into six stages. The results showed that the optimal concentrations for MS-222 for anesthetizing yellowtail kingfish were 100~120 mg/L and 100 mg/L at water temperatures of 20℃ and 24℃, respectively. For clove oil, the optimal concentration for anesthetizing yellowtail kingfish was 40 mg/L at both temperatures. All the experimental yellowtail kingfish were anaesthetized within three minutes and recovered within five minutes. By increasing the anesthetic concentration, the time for the yellowtail kingfish to enter anesthesia was reduced and the recovery time prolonged. The increase in water temperature reduced the time to enter anesthesia, but had no obvious effect on the recovery time. When anesthetized with 40 mg/L clove oil at 20℃ and 24℃, serum cortisol significantly peaked at 6 h and 12 h, respectively (P<0.05). At 24℃, the serum adrenaline level significantly peaked at 12 h (P<0.05) in yellowtail kingfish anesthetized with 100 mg/L of MS-222. The levels of serum adrenaline, glucose, and cortisol in the other experimental groups all significantly peaked after 24 h (P<0.05) in fish anesthetized using both anesthetics. Under the two temperature conditions, the two anesthetics caused the three serum hormone levels to all decrease significantly (P<0.05) to low levels that were lower than the initial levels after 72 h of recovery, indicating that yellowtail kingfish could be physiologically adaptive to the optimal treatment methods of clove oil and MS-222 under different temperatures. The results from the present study provide theoretical and technical support for the development of standard experimental and farming management technology for yellowtail kingfish.