Abstract:Due to the features of maternal inheritance, simple structure, conserved organization, small genome size, and high mutation rate, the mitochondrial genome (mitogenome) has been widely employed in population genetics, comparative genomics, and phylogenetic studies. The body type of Anomura (Crustacea: Decapoda) is between shrimps and crabs, which is of great significance in the study of system evolution. Compared with Brachyra, the closest relative, there has been a significant lack of attention to the study of Anomuran mitogenomes. So far, the mitogenome and gene rearrangement of this group have not been systematically and comprehensively understood. In this study, the research history and development on Anmouran mitogenomes have been briefly reviewed. We compared 26 Anomuran mitogenomes published in GenBank and summarized the fundamental features of these mitogenomes. The common rearrangement types and possible mechanisms of Anomuran mitogenomes were further analyzed. Using the ancestral gene arrangement of crustaceans as a reference, we summarized the mitogenomes of 26 Anomuran species into 15 rearrangement patterns. Gene rearrangement analysis of these mitogenomes showed that only two types of rearrangements were found, including translocation and inversion. Besides, we found that all these rearrangements can be reasonably explained by two rearrangement mechanisms, tandem duplication/random loss and intramitochondrial recombination. Finally, the application of rearrangement in the phylogeny of this taxon was discussed.

Abstract:China is the world's largest producer and consumer of aquatic products, with aquaculture production ranking first in the world for more than 20 consecutive years, and the demand for aquatic products provides opportunities for the development of the global aquaculture industry. In aquaculture, the aquaculture water environment provides the living environment, food and oxygen for freshwater or seawater. Due to human activities, environmental pollution, agricultural production and other reasons, it may lead to changes in total phosphorus, dissolved oxygen, pH and other indicators in aquaculture waters, which in turn affect the growth of aquatic organisms. Therefore, real-time monitoring and prediction of water quality parameters is an important part of the aquaculture process and is an important measure to determine the quality of aquatic products. Through the analysis of the collected information, there are more neural network research results, which play an important role in accurate water quality prediction, but the lack of scientific classification in the literature and the low usage rate of the literature have made it difficult for scholars to find the research entry point. To address this issue, this paper classified the literature on neural networks methods for accurate prediction of farmed water quality according to two major fields: seawater and freshwater, and mainly studied and analyzed the neural network models applied in each field for prediction of seawater spatio-temporal sequences from three architectures: positive feedback architecture, recurrent architecture and hybrid architecture, and the analysis results showed that the highest prediction performance in the positive feedback architecture model is the ANN prediction model with 64% accuracy, and in the recurrent architecture model, the highest prediction performance is the convolutional neural network prediction model with 97.1% accuracy, and in the hybrid architecture model, the highest prediction accuracy is the intelligent algorithm-LSTM -RNNs model with an accuracy of 99.72%, which is 35.72% and 2.62% higher than the highest accuracy in the positive feedback architecture model and the recurrent architecture model, respectively. The prediction performance of the hybrid architecture model is better than those of the positive feedback model and the recurrent architecture model, which is conducive to improving the prediction accuracy of the different depth water quality prediction models. In addition, this paper had a preliminary discussion on the three-dimensional water quality prediction model based on the neural network method, and the results showed that the research scholars results are more focused on the changes of water quality parameters in different locations of the water surface layer and water intermediate layer, while for neural network prediction model for water surface layer, water quality prediction accuracy was higher than intermediate and deep water layer quality prediction accuracy.

Abstract:Interleukin-17 (IL-17) is an important inflammatory factor, which is able to promote the production of various inflammation-related factors, such as IL-1β, IFN-γ, IL-6, CCL20 and so forth. It plays an important role in autoimmune disease, host defense, inflammation and so on. In order to study the pro-inflammatory function of IL-17B in the Cyprinus carpio, the IL-17B gene was cloned, the NusA-17B protein was recombinantly expressed by Escherichia coli system and the pro-inflammatory effect of the IL-17B was then investigated by in vivo and in vitro experiments. In this study, by homology search and gene cloning method, we found two IL-17Bs in the genome of the C. carpio, CcIL-17B1 and CcIL-17B2 respectively. Both of the two genes have open reading frames (ORF) of 597 bp, which are composed of three exons and two introns. The length of their exons are the same, 24, 320 and 253 bp respectively. While the introns are different, the CcIL-17B1 intron are 84 , 420 bp respectively and the CcIL-17B2 intron are 81, 851 bp respectively. The two CcIL-17Bs encode 198 amino acids, containing 2 disulfide bonds formed by 4 cysteines, which were unique to the IL-17 family. Sequence blast analysis showed that the consistency of the two CcIL-17Bs proteins reached 91.92%. The collinearity analysis results are as follows. During the doubling of the chromosomes of teleost, IL-17B and its nearby genes were lost. Most teleost have one IL-17B and Danio rerio have both IL-17Bs that were lost. The common carp-specific chromosomes doubled resulting in the presence of two CcIL-17Bs. Real time quantitative PCR (qPCR) was used to measure the expression of CcIL-17B1 and CcIL-17B2 in fertilized eggs, larvae and different tissues of adult C. carpio. The results showed that the expression levels of both CcIL-17B1 and CcIL-17B2 were significantly higher at 0-12 hours post fertilization than that at 25-90 hours post fertilization (P<0.05). While the expression levels have no significant difference at 25-90 hours after fertilization and 1-14 days after hatching (P>0.05). In adult fish, the expression level of two genes in testis and gonad are significantly higher than that in other tissues, such as spleen, heart, kidney, head kidney, intestine, gill, liver, skin, muscle, brain (P<0.05). Further, using the E. coli expression system, the soluble recombinant protein NusA-17B was obtained. Different concentrations of NusA-17B were injected into the anus to evaluate its pro-inflammatory effect. Histopathological results showed that intestinal villi defect, plenty of goblet cells and inflammatory cells appeared in intestines treated with high concentration NusA-17B (500 μg/kg) after 1 and 3 days. Meanwhile, qPCR showed that IL-1β, IFN-γ, IL-6, CCL20 and NF-κB were significantly up-regulated (P<0.05). However, the abnormal changes in intestinal structure and expression of inflammation-related genes were restored to normal level after 7 days. In addition, in vitro, the C. carpio kidney cells were incubated into different concentrations (0.1, 1.0, 10.0 and 100.0 ng/mL) NusA-17B for 8 hours. The results showed that IL-1β was significantly up-regulated under NusA-17B stimulation at 1.0, 10. and 100.0 ng/mL (P<0.05); IFN-γ and NF-κB were significantly up-regulated under the stimulation of NusA-17B at 10.0 ng/mL and 0.1 ng/mL respectively (P<0.05); IL-6 and CCL20 were significantly expressed under NusA-17B stimulation at 10.0 and 100.0 ng/mL (P<0.05), and TRAF6 was significantly expressed under NusA-17B stimulation at 0.1, 1.0 and 10.0 ng/mL (P<0.05). In summary, the results of in vivo and in vitro experiments show that CcIL-17B participates in the inflammatory response.

Abstract:The ncc gene encodes Na⁺-Cl^? synergistic transporter, while the nkcc gene encodes Na⁺-K⁺-2Cl^?synergistic transporter. Both of them belong to the solute carrier family12 (SLC12), and play key roles in the osmotic regulation mechanism of fish. In order to explore the roles of ncc and nkcc in the osmotic regulation of spotted sea bass in this study, whole genome identification, multiple sequence alignment, phylogenetic tree construction and protein structure prediction were performed to identify the ncc genes of spotted sea bass and analyse their sequence structure. The expression levels of ncc and nkcc genes in gill tissues were detected by qRT-PCR, and the sites of expression of ncc2 and nkcc1a in gills of spotted sea bass in freshwater and seawater were determined by in situ hybridization (ISH). The results showed that two ncc genes, ncc1 and ncc2, were identified in spotted sea bass. Their CDSs length were 2 691 and 3 120 bp, encoding 896 and 1 039 amino acids, respectively. The expression of ncc2 gene in gill tissue of freshwater fish was significantly higher than that in seawater, while the expression of nkcc1a in gill tissue of seawater fish was significantly higher than that in freshwater. Moreover, the expression of ncc2 in gill was gradually up-regulated during freshwater adaptation, while the expression of nkcc1a was gradually down-regulated. Meanwhile, the opposite expression trend was showed in seawater adaptation process. In addition, the ISH results showed that ncc2 and nkcc1a genes were located in the epithelium between adjacent gill lamellae in freshwater and seawater, respectively. The above results indicate that ncc2 and nkcc1a genes encode important Na⁺-Cl^? transporter subtypes in the gills of spotted sea bass in freshwater and seawater, respectively. They can be used as specific markers to distinguish the spotted sea bass in freshwater and seawater, and play an important role in osmotic regulation and salinity adaptation.

Abstract:Cobia (Rachycentron canadum) is an increasingly important marine fish with significant potential for aquaculture in China. Several studies have shown that this fish exhibits distinct locomotion patterns, feeding behaviors and nutritional demands during the larvae and juvenile stages. In order to explore the developmental characteristics of the spine and appendage bones in the early stages of cobia, this paper described the morphologic ossification characteristics of the vertebrae and appendages of larval and juvenile cobia [1 to 33 days post hatching (dph)] using a cartilage bone clearing and staining technique to obtain information on the structural development and functional adaptation of larvae and juveniles organs. After cartilage and bone were stained with alcian blue and alizarin red, vertebral columns were observed to develop from haemal arches and neural arches at 7 dph. Then, the centrum, haemal arches, and neural arches began to ossify at 13 dph. The dorsal rib and ventral rib began to ossify at 17 and 20 dph, respectively, and the vertebrae were fully ossified at 29 dph. The appendicular skeleton developed in the following order: the pectoral fin is the first to develop, followed by the caudal, ventral, dorsal, and anal fins. The pectoral fin began to develop from the cleithrum at 4 dph. The supracleithrum of the pectoral fin ossified at 12 dph and at the same time, the scapula foramen appeared, while the coracoid and scapula began to ossify at 20 dph. The hypural 1 of the caudal fin appeared at 15 dph, the urostyle, parhypural, and fin ray began to ossify at 15 dph, whereas the hypural began to ossify at 18 dph. The ventral fin plate extended to the cleithrum at 17 dph and simultaneously, the ventral fin began to ossify. At 17 and 18 dph, the dorsal and anal fins were ossified in the same pattern. The appendicular skeleton, with the exception of the urostyle, cleithrum, and pelvic girdle, underwent cartilaginous ossification. The development of the vertebral column and appendicular skeleton, on the other hand, is accompanied by changes in feeding patterns and locomotion patterns, such as the emergence of pectoral fin primordia that created conditions for the beginning of larval movement, the development of tail fin folds that enhanced its swimming ability, and the development of pectoral and tail fins in furtherance of cobia’s quick swimming. The results of the study show that exercise skeletons were preferentially developed, such as ossification of the vertebral column and the appendicular skeleton, which enhanced cobia’s forced swimming ability. In addition, hypural healing has been found during skeletal development. In accordance with this process, this paper considered that the morphotype transition of the caudal fin is associated with hypural healing. It is suggested that the osteological ontogenesis of larval and juvenile cobia is closely related to changes in their movement patterns. At 22 dph, scale outgrowth could be found on the caudal peduncle, the vertebral column and appendicular skeleton tended to be fully ossified, and the body features of the fish are the same as adult fish except for the shape of the caudal fin. This study proposes the use of the start of vertebrae ossification as a criterion to distinguish between larval and juvenile cobia, which are considered to enter the juvenile stage at 13 dph. The results suggest that the early bone developmental characteristics of cobia larvae and juveniles are closely related to their functional adaptation. Research on the development of the vertebral column and the appendicular skeleton of cobia is important to enhance the understanding of environmental preferences and functional morphology.

Abstract:Sepia esculenta belongs to Cephalopoda, Sepioidea, Sepiidae, and Sepia, which has become an essential economic seafood species along the northern coast of China. The beak is the main feeding organ of cephalopods, its morphology and internal structure can record biological information on cephalopods at different stages of growth. In addition, there are differences in the feeding objects of cuttlefish at different stages of individual development, which may affect the morphology of beaks. In order to investigate the effects of ontogeny and feeding habits on the beaks of cuttlefish, 138 pairs of beaks of cuttlefish were collected from the southern Yellow Sea from November 2018 to March 2019. Using the landmark point method of geometric morphometry, the upper and lower beaks images were defined and analyzed by 27 marks. The results showed that ① The upper beaks are slightly larger than the lower beaks. And the transition from immature to maturing may be a transgressive period in the growth of the beaks. ② There were significant differences in the size, morphology, and allometric growth pattern of upper and lower beaks in different developmental stages (P<0.05), and there were significant differences in the morphology of upper beaks in different genders (P<0.05), but lower beaks were not the case (P>0.05). ③ The female upper beaks have a high morphological diversity in the mature stage, while the male upper beaks show this in the immature stage. And the maturing stage has a greater influence on the lower beaks. ④ In the immature stage, the beak of cuttlefish was curved and sharp, which facilitated a quick bite on small amphipods. While in maturing stage, the beak’s hood and wing became wider and the beak’s rostrum was sharp, which helped cuttlefish to capture prey precisely of the fast-swimming and agile fish. However, in the mature stage, the beak’s rostrum was short and blunt, the lateral wall was longer and the beak’s wing was wide, which provided a large bite force to crush the hard shell of crustaceans. The development mechanism of beaks at different developmental stages may be a response to better adapt to the changes of feeding objects and satisfy feeding needs. In addition, environmental factors in the southern Yellow Sea may also lead to morphological changes in the beaks of the cuttlefish. Future studies should focus on the internal mechanism of beak deformation in response to the transformation of feeding objects and environmental change. These are associated with individual growth and feeding habit of phenotypic plasticity reflecting the cuttlefish beak growth regularity and the adaptive use of food resources of cuttlefish. This study, through the analysis of the growth of cuttlefish’s beak shape change, improved the basic biology of cuttlefish information, and may provide a certain scientific basis for reasonable use of resources.

Abstract:In order to develop methylation markers related to low salinity tolerance in Portunus trituberculatus, in this study, methylation sensitive high resolution melting curve method (MS-HRM) was applied to screen and validate methylation loci related to low salinity tolerance. A total of 8 methylation markers were developed from the transcriptome database of P. trituberculatus, six of which were significantly related to low salinity tolerance, exhibiting a significant demethylation or methylation pattern in the low salinity condition. They were located in V-type proton ATPase, H⁺/Cl^? exchange transporter, succinate dehydrogenase, NAD(P) transhydrogenase, phosphoglycolate phosphatase, NADH dehydrogenase genes, and these markers provided useful tools for marker-assisted breeding of low-salinity-tolerant strain of P. trituberculatus. The low salinity tolerance test and genotyping results of these 6 loci in wild populations and “Ningxiang-1” selective strain showed that the survival rate of “Ningxiang-1” was significantly higher than that of wild populations under low salinity conditions, and the demethylation of loci Pt-M1 and Pt-M2 in the selective breeding population was significantly increased to 100%, which may be beneficial to maintaining the balance of Na⁺ and Cl^? under low salinity conditions. This study provides basic data and favorable tools for genetic improvement of low salinity resistance in P. trituberculatus.

Abstract:In order to isolate the brain microvascular endothelial cells(BMECs) from Pelophylax nigromaculata, the brain tissue was used in this study. Under an asepic condition, the cerebral cortex was digested with 0.1% collagenase type II and collagenase/dispase, then separated by Percoll density gradient centrifugation. BMECs were inoculated in culture flask coated with rat tail glue, and cultured at 28 °C, 5% CO₂. Then, the cells were identified by morphologic observation and immunofluorescence staining with factor Ⅷ. Besides, the methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to detect the cell viability. The results showed that after 1 day, the microvascular fragments grew adherently and proliferated in a clustered monolayer, then expanded in culture and formed confluent monolayers with the characteristic cobblestone shape on 7th day. Positive Ⅷ expression was frequently observed in P. nigromaculata BMECs cytoplasm. Besides, MTT assay showed the cell viability of P. nigromaculata BMECs grew the fastest on the 3rd day to the 5th. In this study, an available method of culturing P. nigromaculata BMECs was established for the first time, which is of great significance for studying the pathogenesis of frog neurological diseases in vitro, providing a cell model for the development and screening of related drugs.

Abstract:As a pelagic shark species, the silky shark, Carcharhinus falciformis, in the Eastern Pacific has been overfished in the tuna purse-seine and longline fishery and requires improved assessment to enable planning of recovery actions. The present study examined the life history of 802 silky sharks (67 individuals were sampled, 35 females and 32 males) from the Eastern Pacific. Age was analyzed using sectioned vertebrae, and a multimodel approach was applied to the length-at-age data to fit growth models. Silky sharks ranged in dominant length from 160 to 220 cm total length (L), while ranged in dominant age from 9 to 13 years. Females the longest was 222.58 cm total length, with the oldest estimated at 18 years. Males the longest was 233.47 cm total length, with the oldest estimated at 17 years. The Logistic growth model provided the best fitting growth model. Parameter estimates for females were: asymptotic length (L_∞) = 231.51 cm; growth coefficient (g) = 0.19/a; and length at age 0 (L₀) = 68.89 cm. For males, the parameter estimates were: (L_∞) = 251.96 cm; (g) = 0.15/a; and (L₀) = 68.71 cm. Females matured at (13.50±0.43) years and (207.60±2.43) cm, whereas males mature at (13.42±0.46) years and (200.67±3.60) cm. The growth parameters and late ages of sexual maturation for silky sharks in the Eastern Pacific suggest a significant risk from fisheries exploitation without careful population management.

Abstract:Yellow catfish (Pelteobagrus fulvidraco) has good taste and nutritional value, and can provide the necessary mineral elements for the human body. In order to explore the content of mineral elements in different tissues, the experiment use the P. fulvidraco as the object. The distribution and content of calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), manganese (Mn) and selenium (Se) were determined by flame atomic absorption spectrometry in 18 tissues of P. fulvidraco (heart, liver, brain, spleen, kidney, muscle, fat, fore-intestine, mid-intestine, eyes, gill, tail fin, swim bladder, bone, stomach, skin, blood and whole fish). The results showed that Ca accounted for a higher proportion in bone, gill, tail fin and muscle, and the Ca content was the highest in tail fin and bone and the lowest in fat; Mg accounted for a higher proportion in bone and muscle, and the Mg content was highest in bone and tail fin and the lowest in eyes; Zn had a higher proportion in the bone, muscle, skin and eye, and the Zn content was the highest in eyes and the lowest in fat tissue; Fe had a higher proportion in the bone, blood and muscle, and the Fe content was the highest in blood and the lowest in fat; Cu had a higher proportion in bone, muscle and liver, and the Cu content was the highest in kidney and liver, while the lowest in fat and eyes; Mn had a higher proportion in bone and muscle, and the Mn content was the highest in bone and tail fin and the lowest in eyes and fat; Se had a higher proportion in muscle, bone, skin, and liver, and the Se content was highest in spleen, liver, kidney, and fore-intestine, and the lowest in muscle and fat; The present study used ICP-MS to explore the distribution and content of mineral elements (Ca, Mg, Zn, Fe, Cu, Mn and Se) in different tissues of P. fulvidraco, which provided good basis for determining the nutritional value of mineral elements in P. fulvidraco, and also has important significance for related research of mineral elements in other fishes.

Abstract:Mytilus coruscus is an important economic shellfish and a typical marine macrofouling organism in China. The process of settlement and metamorphosis in its life history is a necessary condition for the survival of larvae. The effects of cellulose on the biofilms of Pseudoalteromonas marina biological characteristics and its effect on the larval settlement and metamorphosis of M. coruscus were explored. Biofilms were cultured by different concentrations of cellulose (0.02, 0.2, 2 and 20 mg/L) with P. marina (initial bacterial density: 5×10⁸ cells/mL). The inducing activity of biofilms formed under different conditions was tested. The results showed, when P. marina biofilm formed with the cellulose concentration of 2 mg/L, the percentage of larval settlement and metamorphosis was 25%, which was significantly lower than that of monospecific bacterial biofilm. As the cellulose concentration increased, the bacterial density of the biofilm formed by P. marina and cellulose was greatly reduced, and reached the minimum at a certain concentration (20 mg/L). The bacteria were more dispersed and biofilm thickness were significantly reduced. Compared with monospecific bacterial biofilm, the biomass of α-polysaccharides, β-polysaccharides and lipids of biofilms formed with addition of cellulose was reduced by 72.55%, 62.01% and 66.23%, respectively. While for proteins, no significant difference was observed in the presence or absence of cellulose. In the process of P. marina biofilm formation, cellulose indirectly regulated the larval settlement and metamorphosis by reducing the production of extracellular polysaccharides and lipids. The findings may provide a theoretical basis for exploring the molecular mechanism of P. marina cellulose on the formation of biofilms and the settlement and metamorphosis of M. coruscus, and also provide new ideas for the treatment of biofouling.

Abstract:Sea cucumber Apostichopus japonicus is one of most valuable marine cultured species in northeast China, and the cultured production reached 170 000 tons in 2020. With the rapid development of A. japonicus breeding industry, high efficiency and environmental protection formula diet has become one of bottleneck problem which inhibited the development of industry. Vitamin A (V_A), also called retinol, is the general name of monounsaturated alcohol or alcohol active substances, which is an extremely important and easily deficient fat soluble vitamin. V_A can maintain vision, promote cell differentiation and bone development, and participate in fish mucus secretion and fat metabolism. Meanwhile, it also can improve animal growth rate, reproductive performance and disease resistance of Cyprinus carpio var. Jian and Scophthalmus maximus, but long term excessive intake of V_A may result in bone deformities of Sparus aurata. So it is very important to determine the optimum dietary V_A in formulate diet. In order to investigate the effects of dietary V_A on growth performance, body composition, digestive metabolism and non-specific immunity for A. japonicus juveniles, an 8-week feeding experiment was conducted with A. japonicus juveniles. Six isonitrogen and isoenergetic diets with graded levels V_A[3 250(D1), 5 187(D2), 7 054(D3), 8 970(D4), 12 975(D5), 16 400(D6) IU/kg] were formulated by adding V_A acetate into basal diet. Each diet was assigned to triplicate tanks with 30 A. japonicus juveniles with initial body weight (15.48±0.01) g. Results showed that there was no significant effect on survival rate, but both the weight gain rate and specific growth rate were significantly increased. With the increasing of dietary V_A levels, the contents of crude lipid and V_A of body wall were increased, and crude ash contents increased firstly and thereafter declined. The contents of hydroxyproline were decreased and D1 group was significantly higher than other groups. The activity of lipase was increased with the increasing of dietary V_A levels, and D1group was significantly lower than other groups. The activity of amylase was increased first and then decreased afterwards. There were no differences in protease activities among all groups. With the increasing of dietary V_A levels, all of the activities of aspartate aminotransferase (AST), total superoxide dismutase (T-SOD) and catalase (CAT) were first elevated and then fell down, and the activities of AST and CAT reached the highest in D4 group. The activity of T-SOD of D3 and D4 groups were significantly higher than other groups. The activity of alkaline phosphatase showed an upward trend and D1 group was significantly lower than other groups. The contents of malondialdehyde were decreased. Taking the weight gain rate as the evaluation index, analysis by a linear regression equation indicated that the optimum dietary V_A requirement for A. japonicus (initial body weight 15.41g) was 10 000 IU/kg. The results of this experiment can provide a reference for the addition of V_A in the formula feed, and also provide a basis for the development of "micronutrient balance" formula feed of A. japonicus.

Abstract:Schizothorax macropogon is one of the main economic fishes in Tibet area, only distributed in the upper and middle reaches of the Yarlung Tsangpo River and its tributaries. Saprolegniasis is one of the most difficult fish diseases in prevention and control, and brings huge economic losses to the aquaculture industry. This experiment was conducted to analyze the effects of Saprolegniasis on the spleen transcriptome of S. macropogon and understand the molecular mechanisms of S. macropogon infected by Saprolegniasis. A total of 5 healthy and 5 Saprolegnia infected S. macropogon living in the same aquatorium were randomly selected. The PDA agar medium and molecular biology methods were used for isolation and identification the S. strain from S. Macropogon. In addition to the transcriptomes of spleen tissues of healthy and fungus infection, S. macropogon were sequenced based on the Illumina HiSeq^TM 2000 high-throughput sequencing platform and the data were analyzed by bioinformatics tools in this study. The research showed that we obtained 3 S. strains from the skin of S. Macropogon, including S. parasitica and S. ferax. A total of 46 619 504 and 43 912 876 clean reads were obtained in the healthy group and fungus infection group, respectively. 359 164 transcripts were assembled and 123 863 unigenes were spliced after removing redundancy. Compared with the healthy group, 1 889 unigenes showed differential expressions in the fungus infection group, 1 414 unigenes were up-regulated and 475 unigenes were down-regulated among these genes. Six differentially expressed unigenes (natural killer cells-lysin gene, somatostatin gene, preproinsulin gene, chymotrypsin gene, protein disulfide isomerase gene and prolactin receptor gene) were randomly selected for quantitative real-time PCR and the results were consistent with the RNA-seq. The results of GO functional enrichment showed that the up-regulated genes are mainly enriched in 241 terms, and down-regulated genes are mainly enriched in 60 terms. These genes involve molecular functions, cell components and biological processes. Besides, among these GO functions, the most differentially expressed genes were enrichment in biological processes, including muscle contraction, muscle system process and muscle structure development. KEGG pathway enrichment suggested that the unigenes were enriched in immune diseases, endocrine and metabolic diseases, viral infectious diseases, digestive system, excretory system and so on. Collectively, the results of above indicate that infection with Saprolegnia can affect genes expression in the spleen tissue of S. macropogon. The conclusion will provide important references for further exploration of the S. infection mechanism of S. macropogon.

Abstract:The aim of this study was to explore the function and regulatory mechanism of tumor necrosis factor receptor-associated factor 3 (TRAF3) in fish antiviral immune response. In the present study, the transcript encoding TRAF3 (AjTRAF3) was obtained from Japanese eel, Anguilla japonica, by using reverse transcription PCR. The structural characteristics of AjTRAF3 were analyzed using bioinformatics softwares and its expression profile and functional mechanism were investigated by qPCR, luciferase reporter and co-immunoprecipitation assay. The open reading frame of AjTRAF3 was 1 707 bp long, encoding a polypeptide consisting of an N-terminal ring domain, two Zn Finger domains, a helical domain, and a C-terminal TRAF-C (MATH) domain. qPCR analysis revealed a wide tissue distribution of AjTRAF3, with the highest expression in brain, followed by head kidney, and the lowest in heart. The highest induction of AjTRAF3 in response to Poly I:C stimulation was observed at 24 hour post injection (hpi) in spleen, being 15.83 fold higher than control. The highest up-regulation of AjTRAF3 after E. tarda infection was observed in spleen at 24 hpi, being 31.47 fold higher than control. Furthermore, the eukaryotic expression plasmid was constructed to study the function of AjTRAF3 in vitro. Our results revealed increased expression of antiviral related genes and increased luciferase transactivation of the AjIFN2, AjIFN4 and NF-κB promoter in cells overexpressed with AjTRAF3. Further, AjRIG-IN, AjMAVS- or AjIRF3-induced AjIFN2, AjIFN4 and NF-κB promoter activation was significantly enhanced in cells co-transfected with AjTRAF3. In addition, subcellular localization analysis revealed the cytoplasmic distribution of AjTRAF3 and the co-localization of AjTRAF3 with MAVS on mitochondria. Lastly, co-immunoprecipitation assays showed that AjTRAF3 interacts with AjMAVS via the MATH domain, whereas a mutant lacking this domain lost the ability to interact with AjMAVS. Collectively, these data suggested that AjTRAF3 can induce antiviral responses by regulating RIG-I/MAVS-mediated signaling. These findings contribute to understanding of function of AjTRAF3 in antiviral response.

Abstract:Ranavirus has a wide range of hosts, can infect reptiles, fish and amphibians, and lead to a high mortality. In order to further enrich its detection methods, in this study, a pair of specific primers was designed for the frog virus type 3 (FV3) based on its major capsid protein gene, and two other frog virus PCR detection methods were selected for comparison experiments. After the optimization of PCR reaction system, specificity and sensitivity test, a new PCR method for the detection of FV3 strain was established. This method has a minimum detection limit of 1.2 copies, has no cross-reaction with nervous necrosis virus, shrimp hemocyte iridovirus, largemouth bass Ranavirus, koi herpesvirus, carp edema virus, infectious spleen and kidney necrosis virus and Micropterus salmoides rhabdovirus. The present new method was used to detect the clinical samples, obtaining consistent results with the other two methods. The FV3 PCR detection method established in this research has the characteristics of simplicity, rapidity, good sensitivity, high specificity, and low cost. It can be used for rapid diagnosis and molecular epidemiological investigation of FV3 frog virus.

Abstract:To investigate the effect of salt amount and salting time on the quality attributes of roasted fillets from Ctenopharyngodon idella, the scales, head, tail, and internal organs of fresh C. idella were removed and cut into fillets, then salted respectively using a dry salting method [salt amount (5%, 6%, 7%) for 4 h, or salting time (2, 4, 6 h) with 6% of salt] and roasted at 200 °C for 15 min. The salt content, weight loss rate, and physicochemical properties of myofibrillar protein [secondary structure, surface hydrophobicity index (H₀), and total sulfhydryl and disulfide bond contents] of the salted fillets from C. idella, as well as the shear force, color, sensory score, and moisture, ash, and protein contents of roasted fillets from C. idella were evaluated. The results showed that as salt amount increased and salting time prolonged, the contents of salt and disulfide bond and H₀ of the salted fillets from C. idella gradually increased, the total sulfhydryl content underwent an opposite trend. α-helix and β-sheet of myofibrillar protein were turned into β-turn and random coil. Compared to the control (unsalted), the L^* and a^* values, shear force, and moisture and protein contents of the roasted fillets from C. idella with salting dropped sharply, while the ash content significantly increased. Furthermore, as the salt amount increased or salting time prolonged, the moisture content increased and ash content decreased, while the sensory score firstly increased, followed by decreasing. These results suggested that salt amount added and salting time significantly affected the secondary structure of the myofibrillar protein from salted C. idella fillets, and enhanced their denaturation, leading to the changes in quality attributes of the roasted fillets from C. idella. Compared to other salting conditions, the roasted fillets from C. idella had higher moisture and protein contents and lower ash content, as well as the better color and tenderness and the higher sensory score when fillets were salted for 4 h at 6% of salt.

Abstract:Selenium is one of the essential trace elements in the human body. Lack of selenium can lead to many diseases, such as Keshan disease, large bone disease and so on. Compared with inorganic selenium, organic selenium can cross the intestinal wall actively, so it is necessary to develop organic selenium with high biological activity. Selenium chelate, a collagen protease hydrolysate, has good antioxidant properties, can also supplement selenium and collagen for human body, and has high bioavailability. In order to make efficient use of tilapia by-product resources and provide theoretical basis for the industrialization of selenium chelate, tilapia skin collagen (TSC) was used as raw material for enzymatic hydrolysis by 4 kinds of proteases. The enzymolysis products of acid protease (TSCAc), papain enzymolysis product (TSCP), bromelain enzymolysis product (TSCB) and alkaline protease enzymolysis product (TSCAl) were obtained. Using Na₂SeO₃ as the source of selenium, the hydrolysate with the highest selenium binding amount was prepared and screened. UV, fluorescence, FTIR and surface hydrophobicity of the enzyme hydrolysate and its selenium chelate were determined, and the antioxidant activity and antioxidant stability of the enzyme hydrolysate and its selenium chelate were analyzed. The hydrolysate of alkaline protease (TSCAl) had the highest hydrolysis degree (18.2%), and the proportion of molecular weight < 1 000 u was 53.7%, which was higher than that of other hydrolysates. The chelate of alkaline protease (TSCAl-Se) had the highest selenium binding amount (153.5 mg/g) and the selenium binding rate was 23.7%. After the chelation of TSCAl and selenium, the intensity of the absorption band at 210-238 nm wavelength decreased and showed a blue shift, and the intensity of the absorption band at 238 nm-300 nm wavelength increased. After the chelation of TSCAl and selenium, the fluorescence intensity of the fluorescence spectrum weakened, and the absorption peak red shifted 2 nm. After TSCAl chelated with selenium, in the infrared spectrum, the absorption peak of N-H moved from 3 280 cm^?1 to 3 365 cm^?1, the absorption peak of O-H shifted from 1 393 cm^?1 to 1 400 cm^?1, another absorption peak of O-H moved from 1 446 cm^?1 to 1 456 cm^?1, the absorption peak of C-H shifted from 2 929 cm^?1 to 2 930 cm^?1, the absorption peak of C=O shifted from 1 635 cm^?1 to 1 652 cm^?1. These indicate that TSCAl and selenium combine to form a new substance. The IC₅₀ values of TSCAl, TSCAl-Se and Na₂SeO₃ for scavenging OH radicals were 6.6 mg/mL, 0.05 mg/mL and 0.17 mg/mL, respectively. The antioxidant activity of TSCAl and TSCAl-Se fluctuated slightly at different temperature but was relatively stable. The antioxidant stability of them decreased at 100 °C. With the increase of pH, the antioxidant activity slowly increased first and then decreased, and reached the highest value at pH=7. These results indicate that the selenium chelate of alkaline protease hydrolysate of tilapia skin collagen has good antioxidant activity and antioxidant stability, which provides a theoretical basis for the research and development of selenium dietary supplements and antioxidant.

Abstract:Lutein is a highly effective antioxidant, but its application is limited because its stability is easily affected by the environment. Tilapia protein isolate (TPI) is a high-quality animal protein with balanced amino acid ratio and high digestibility. However, due to the limitation of low solubility in water, its emulsifying activity and emulsifying stability are poor, and the application of TPI as emulsifier is limited. The pH value is the most obvious external factor that affects the functional properties of protein, and heat treatment is an essential operation in food processing. In order to improve the stability and bioavailability of lutein, tilapia protein isolate (TPI) was used as emulsifier, and TPI emulsions containing 200 μg/mL lutein were prepared by high pressure homogenization to explore the effects of heat treatment (70 °C, 30 min) on emulsion particle size, creaming index, lutein content, in vitro antioxidant activity and in vitro digestion at three pH values (3.0, 7.0 and 10.0). The results showed that after heat treatment, the particle size of TPI emulsion with lutein decreased, the emulsion droplets were evenly distributed, and the storage stability was enhanced. At pH 3.0, the stability of TPI emulsion loaded lutein was poor with lutein degradation, and the color of emulsion changed obviously. However, at pH 7.0 and 10.0, heat treatment of 70 °C significantly improved the stability of the emulsion without causing the degradation of lutein, and there was no delamination when stored at 4 °C for 28 days. After simulated gastrointestinal digestion in vitro, the release rate of free fatty acids was accelerated. After heat treatment, the bioavailability of lutein increased from 18.65%±1.06% to 35.69%±2.06% at pH 7.0. At pH 7.0, the free fatty acid release was 94.22%±0.60%, the bioavailability was 35.69%±2.06%, and the ATBS free radical scavenging ability was 59.17%±0.66%. The results showed that combined heat treatment under neutral and alkaline conditions can enhance the stability and bioavailability of lutein loaded TPI emulsion and protect the antioxidant activity of lutein, which provides a basis for the development and application of lutein-loaded TPI emulsion.