Abstract:Crustaceans and insects are close in evolution and referred to as pancrustaceans. Most vitellogenin is a female-specific protein, which is the precursor of vitellin stored in oocytes. Vitellogenin plays a key role in reproductive success. It has been known that juvenile hormone, molting hormone, neuropeptide and insulin-like peptide are the main factors for regulating vitellogenin synthesis in pancrustaceans. The juvenile hormone and ecdysone exert promoting effect on vitellogenesis in insects, while the effect of neuropeptides and insulin-like peptides on the vitellogenesis in a species-specific manner. In addition to the above hormones, the crustacean unique hormones, such as eyestalk hyperglycemic hormone (CHH) family and insulin-like androgenic gland hormone (IAG), have inhibitory effects on the vitellogenin synthesis. This review summarized the research progress on the regulatory mechanism of vitellogenin synthesis in pancrustaceans, and make detail comparisons of the main hormones and their regulation of vitellogenin synthesis between insects and crustaceans, providing a valuable reference for future studies on reproduction control in cultivated decapod crustaceans.

Abstract:Vitamin D₃ is an essential vitamin for aquatic animals and functions to maintain normal growth, development, and nutritional metabolism. Aquatic animals barely synthesize vitamin D₃ in the body. Instead, they obtain vitamin D₃ mainly from feed. To gain deep knowledge concerning mechanisms by which vitamin D₃ regulates carbohydrate and lipid metabolism in aquatic animals, we summarized dietary vitamin D₃ requirement and its influencing factors, and illustrated vitamin D₃ absorption, transport, metabolism and regulatory mechanisms, and the mechanism of vitamin D₃ regulating carbohydrate and lipid metabolism in aquatic animals. At present, studies are very limited on nutrition physiology, metabolism and regulation on carbohydrate and lipid metabolism by vitamin D₃ in aquatic animals, and many important scientific questions remain unresolved. In the future, it will be necessary to set up a unified standard evaluating “dietary vitamin D₃ requirement of aquatic animals” and to target studies concerning mechanisms regulating carbohydrate and lipid metabolism in aquatic animals, which will provide references for precise regulation of vitamin D₃ nutrition physiology in aquatic animals.

Abstract:C-type lectin receptor (CTLR) is a kind of pattern recognition receptors that can specifically bind to carbohydrates pathogen-associated molecular patterns (PAMPs) and plays an important role in innate immunity. In order to elucidate the biological function of CTLR in teleost fish, a CTLR gene-Clec4e (C-type lectin domain family 4 member E gene, Clec4e), which was screened from the transcriptome database of large yellow croaker (Larimichthys crocea), was taken as the object of the present study, and its molecular features, expression distribution and agglutination characteristics were studied. The results showed that the full-length cDNA of LcClec4e was 1 546 bp, with an open reading frame (ORF) of 771 bp, encoding 254 amino acids. The N-terminus of LcClec4e had a transmembrane region without a signal peptide, and the C-terminus had a carbohydrate recognition domain (CRD), which contained carbohydrate-binding sites EPN and WFD and 6 conserved cysteines what could form disulfide bonds. Phylogenetic analysis showed that LcClec4e was closely related to CTLRs of a variety of fishes of the order Perciformes. The results of real-time quantitative PCR showed that LcClec4e was constitutively distributed in the 10 tissues tested, and the expression level was the highest in the liver; LcClec4e was expressed in primary macrophages, lymphocytes and granulocytes from the head kidney tissues of L. crocea, and the expression level was the highest in macrophages; the expression levels of LcClec4e in these 3 kinds of immune cells were significantly upregulated after stimulation by inactivated Vibrio alginolyticus. The extracellular segment of LcClec4e (rLcClec4e-extracellular domain, rLcClec4e-ex) expressed in Escherichia coli BL21 had Ca²⁺-dependent agglutination activity and could agglutinate mouse, rabbit erythrocytes and 4 Gram-negative bacteria common in aquaculture, such as Aeromonas hydrophila, Pseudomonas plecoglossicida, V. alginolyticus, V. campbellii. D-glucose, D-fructose, D-mannose, D-maltose, α-lactose, and lipopolysaccharide all had inhibitory effects on the agglutination of rLcClec4e-ex toward P. plecoglossicida, suggesting that LcClec4e could bind to carbohydrates of P. plecoglossicida. The results above indicated that LcClec4e might be an PPR which could recognize bacteria by binding to carbohydrates PAMPs, thus involved in the immune defense of L. crocea against bacterial infection.

Abstract:Currently, the majority of studies investigating the effects of Akkermansia muciniphila primarily focus on blood glucose homeostasis, adipose tissue accumulation, intestinal permeability, and body weight regulation. However, these studies are predominantly limited to human and mammalian subjects. The specific mechanism by which A. muciniphila regulates glucose metabolism in fish remains unclear. So we investigate the molecular mechanism of pasteurized A. muciniphila regulating glucose metabolism in Cyprinus carpio. In this study, different sugar concentrations (20%, 30%, 40%, 50% glucose) were examined to investigate the temporal and spatial variations in glucagon-like peptide-1 secretion and A. muciniphila colonization at 4 and 8 weeks of C. carpio (10.5±1.0 g). Based on the different sugar concentration tests, groups with a glucose level of 40% and three varying concentrations of pasteurized A. muciniphila (10⁸, 10⁹, 10¹⁰ CFU/g) were established. The regulation of glucose metabolism and glucagon-like peptide-1 in C. carpio (16.38±0.39 g) after four weeks of pasteurized A. muciniphila supplementation was explored. Additionally, the regulatory mechanism of intestinal A. muciniphila and glucagon-like peptide-1 on carp's glucose metabolism at different glucose levels was investigated through experiments involving primary liver and intestinal cells incubated with pasteurized A. muciniphila. The results demonstrated the following findings: (1) with an increase in dietary glucose content, there was a significant decrease in serum glucagon-like peptide-1b levels (P <0.05). (2) following pasteurized A. muciniphila treatment, there was a significant reduction in serum glucose and glucagon-like peptide-1 levels (P < 0.05). Moreover, there were notable increases in villus height and muscle thickness of the midintestine along with a decrease in glucagon-like peptide-1 content and an increase in short-chain fatty acid content; additionally, muc2 mRNA expression level increased significantly (P<0.05). Furthermore, pasteurized A. muciniphila up-regulated mRNA expression levels of ampk, pi3k, akt as well as glycolytic genes gk and pfk in the liver while down-regulating mRNA expression levels of gluconeogenic gene pepck (P < 0.05). (3) incubation results from primary liver and intestinal cells revealed that after pasteurized A. muciniphila treatment, there was a significant decrease in glucagon-like peptide-1 content within intestinal cells while simultaneously increasing gpr40 mRNA expression; furthermore, hepatocytes showed significantly increased mRNA expressions of glycolysis genes gk and pfk. In conclusion, high glucose diet increases the blood glucose levels, and causes liver and intestinal damage of C. carpio. The addition of exogenous pasteurized A. muciniphila increases the content of short chain fatty acids in C. carpio intestinal contents, inhibits intestinal secretion of glucagon-like peptide-1, alleviates the increase in blood glucose caused by high glucose diet, maintains glucose homeostasis. This research can provide the practical basis for the application of A. muciniphila as a probiotic in aquatic feed.

Abstract:Mesenteric fat weight (MFW) is a representative index used to reflect body lipid level in Ctenopharyngodon idella. However, the traditional measurements by artificial scrape and weighing are time-consuming, labor intensive and having high operating errors, especially when many samples are to be handled. To avoid such limitations, this study developed a rapid and sensitive method for the quantitation of mesenteric fat utilizing the lysochrome dye oil red O. The initial operation procedures included: fish anesthetization, sample collection of visceral mass, PIT-tag implantation, sample fixation, sample dehydration, oil red O staining. After the above steps, mesenteric fat could be specifically stained by oil red O dye, and all the samples were stained uniformly. After sample extraction and absorbance measurement of extraction liquid, the weight of oil red O extracted from the stained sample could be accurately obtained according to the standard curve of oil red O (y = 0.0276x + 0.0403, R² = 0.999 7). The oil red O weights were used as MFWs in this study, and had good consistency with the fat weights quantified by the conventional scraping method (r = 0.80). Furthermore, data analysis showed that the coefficient of variation for MFW (24.49%) was greater than that for body weight (8.93%) in the cultured population of C. idella (n = 200), indicating a significant potential for its genetic improvement. Correlation and cluster analysis showed that MFW was positively correlated with all the observed traits, of which visceral weight (VW) had the highest correlation coefficient of 0.60 (P ? 0.01), and was clustered into one group. The results were as expected that both MFW and VW belonged to viscera-related indexes. Multiple linear regression analysis showed that morphological indexes could explain only a small amount of variation for MFW (R² = 0.20), indicating that the regression prediction was unsatisfactory, and direct measurement remained an effective approach. This study provided an accurate quantitative method for genetic improvement of body lipid traits in C. idella.

Abstract:Phosphorus is an important biogenic element necessary for the growth and reproduction of marine phytoplankton, and it is also the basic element of marine primary productivity and food chain. The potential content of bioavailable phosphorus (BAP) in sediments that can participate in interface exchange depends on the form of phosphorus in sediments, while there are few studies in the central region of the Bohai Sea. In this study, five areas in the central Bohai Sea (Tangshan coast, Qinhuangdao coast, northeastern Bohai Sea, Bohai Strait, and southwestern Bohai Sea) were selected as the research objects. The composition and content of phosphorus in the sediment samples at 23 sampling points were determined by the hierarchical leaching method, and the potential BAP content was calculated. The distribution characteristics and influencing factors were analyzed. The results showed that the inorganic phosphorus (IP) in the surface sediments of the central Bohai Sea was the main form of total phosphorus (TP) in the surface sediments of the central Bohai Sea. The IP contents in the sediments of Tangshan coast, Qinhuangdao coast, northeastern Bohai Sea, Bohai Strait and southwestern Bohai Sea accounted for 78.39%, 79.06%, 71.46%, 84.60% and 81.46% of the TP content, respectively, while the organic phosphorus (OP) accounted for a small proportion. Detrital phosphorus (De-P) was the main occurrence form of IP, accounting for 54.02%, 52.12%, 33.33%, 69.41% and 57.28% of TP on average, respectively. The content of phosphorus in each form of IP was in the order of De-P > Oc-P > Fe/Al-P > Ca-P > Ex-P. River input, sediment grain size and sedimentary environment are the main factors affecting the content and distribution of different forms of phosphorus in surface sediments in the central Bohai Sea. According to the analysis of potential BAP content in sediments, the potential BAP content in the surface sediments of Tangshan coast, Qinhuangdao coast, northeast Bohai Sea and southwest Bohai Sea accounted for 44.77%, 46.94%, 64.87% and 40.54% of TP, respectively, which had strong potential to release phosphorus into water. This study provides a theoretical reference for further study of nutrient cycling and supplement mechanism in the central Bohai Sea and the upper limit of sediment contribution to phosphorus in marine water.

Abstract:Colored dissolved organic matter (CDOM) is an important source of carbon in aquatic ecosystems, and the optical properties of dissolved organic matter (DOM) can be used to trace water masses and provide information about the dynamics of the dissolved organic fraction in seawaters. The ultraviolet-visible absorption spectrum was applied to investigate the chemical composition, sources, spatial distribution characteristics and migration and transformation processes of the CDOM in both the water body and interstitial water in Sanggou Bay, a representative semi-enclosed and mariculture bay in Shandong Province, China. The results indicated that, ① the range of absorption coefficient [a(355)] was 0.23-9.09 /m in Sungo Bay, there are significant differences in the spatial distribution of different water layers, both the surface layer and bottom layer gradually decreased from the nearshore zone to the bivalves & kelp zone, then it rose in the kelp zone and gradually decreased towards the outer sea zone. The a(355) in the kelp zone decreased from surface to bottom and then increased, high-density and large-scale kelp mariculture released a large amount of CDOM into the seawater. The a(355) of sediment interstitial water in each region was 1.3-2.5 times of the sum of the surface, middle and bottom layers, and it was enriched and gradually accumulated in the sediment interstitial water. ② The range of spectral slope (S_275-295) was 0.013-0.036 in Sanggou Bay, the mean values of S_275-295 in outer sea zone and kelp zone were significantly higher than that in other zones, the main components of CDOM are marine organic matter in kelp zone and outer sea zone, and terrigenous organic matter is predominant in the nearshore zone. The content of humic acids in CDOM in the nearshore zone was gradually decreased from the surface layer to the bottom layer, the accumulation of fulvic acid in sediments was high, and kelp zone and outer sea zone showed opposite trend. ③ The range of specific UV absorbance (SUVA₂₅₄)was 4.60-14.10 L/(mg·m) in Sanggou Bay, the SUVA₂₅₄ of the shellfish zone, the bivalves & kelp zone and the kelp zone showed a trend of increasing gradually from the surface to the bottom layer, and the aromaticity of CDOM was gradually increased and reached the maximum in the sediment interstitial water. The SUVA₂₅₄ in the sediment interstitial water of kelp zone and outer sea zone were significantly higher than that in other zones. This study showed that large-scale suspended cultivation activities from kelp zone contributed more refractory dissolved organic matter to the mariculture zone and adjacent seawaters, and it implied that kelp mariculture had a strong carbon sink effect and this sequestration was exported to the offshore waters through the current.

Abstract:Since the non-ionic ammonia and ionic ammonia can be converted into each other, in aquaculture, ammonia nitrogen (NH₄⁺-N) and nitrite nitrogen (NO₂^--N) are the key factors that affect the growth and development of aquatic animals. Therefore, the removal of NH₄⁺-N and NO₂^--N in aquaculture water is of great significance to ensure the health of aquaculture animals. As we all know, microorganisms and algae play important roles in maintaining the ecological balance of aquaculture ponds. It has been proved that Chlorella had the ability of purifying aquaculture water, with different Chlorella species different removal efficiency on NH₄⁺-N and NO₂^--N. Chlorella vulgaris is widely used in aquaculture, however, we know little about the removal effect on nitrogen nutrient especially NO₂^--N by C. vulgaris. Given the shortage of effective ecological control measures in aquaculture, it is of great significance to illustrate the removal effect of NH₄⁺- N and NO₂^--N by C. vulgaris and related influencing factors. Aiming to evaluate the application prospect of C. vulgaris in purifying NH₄⁺-N and NO₂^--N in water, in the present study, C. vulgaris was taken as the research object and feed wastewater was taken as the culture medium. We firstly detected the cell density of C. vulgaris and the temporal variations of NH₄⁺-N and NO₂^--N in water under aeration, light, combined light and aeration conditions. Then we analyzed the effects of time (X₁), light intensity (X₂) or initial C. vulgaris density (X₃) on the removal rates of NH₄⁺-N and NO₂^--N (Y). Finally, we evaluated the removal efficiency of NH₄⁺-N, NO₂^--N and NO₃^--N from water by C. vulgaris, and we analyzed the potential pathway of NO₂^--N assimilation by C. vulgaris. The results showed that C. vulgaris could remove NH₄⁺-N, NO₂^--N and NO₃^--N significantly under suitable light conditions. The NH₄⁺- N removal rate reached up to at 18 000 lx (96.23%), and NO₂^--N removal rate reached up to 99.19% at 9 000 lx. The initial density of C. vulgaris at 2.5×10⁵ cells/mL had the highest removal rates for NH₄⁺-N and NO₂^--N, accounting 94.92% and 99.05%, respectively. The regression equation of NH₄⁺-N and NO₂^--N removal rates with treatment time and light intensity was as follows: Y_NH4⁺-N=1.189X₁+5.79×10^-4X₂+24.158 (R²=0.664), Y_NO2^--N=1.562X₁+1.909×10^-3X₂-26.078 (R²=0.762). The regression equation of NH₄⁺-N and NO₂^--N removal rates with treatment time and initial C. vulgaris density was as follows: Y_NH4⁺-N =0.888X₁+1.02×10^-5X₃+32.555 (R²=0.408), Y_NO2^--N =1.746X₁+1.64×10^-5X₃-17.250 (R²=0.613). The order of nitrogen removal by C. vulgaris was NH₄⁺-N>NO₃^--N>NO₂^--N, and the activity of nitrite reductase in C. vulgaris at NH₄⁺-N decline stage was significantly lower than that at NO₂^--N decline stage. In conclusion, C. vulgaris can significantly reduce the contents of NH₄⁺-N and NO₂^--N in water, and NO₂^--N may be reduced to NH₄⁺-N by intracellular nitrite reductase and assimilated by C. vulgaris. These results provide scientific basis for in-situ bioremediation of aquaculture waters.

Abstract:Many algal pathogens have a broad spectrum of algae-lysing activity. In order to verify whether Vibrio mediterranei 117-T6 (Vm 117-T6) has broad-spectrum algicide properties, this paper observed its algal lysis on Heterosigma akashiwo, Pleurochrysis carterae, Dirateria inornata and Prorocentrum donghaiense, and the characteristics of the algae-lysing substances were analyzed. In this study, Vm 117-T6, a pathogenic bacterium for the yellow spot disease of Pyropia, showed a broad-spectrum algae-lysing activity. It exhibited strong algae-lysing activity to H. akashiwo, P. carterae, D. inornata and P. donghaiense, which significantly reduces the chlorophyll content of these microalgaes. Vm 117-T6 can also reduce the motility of H. akashiwo within 40 min, inhibit photosynthesis, cause algal lysis and produce white flocculent precipitates after infection over the 120 min. Vm 117-T6 can degrade carrageenan, but cannot degrade pectin, cellulose and chitin. The live bacterium, its intracellular and extracellular extracts showed strong microalgae-lysing activity. The extracellular algae-lysing compounds were easily soluble in water, insoluble in petroleum ether and ethyl acetate, which indicated strong polarity. It was resistant to high temperature, and was not easy adsorbed by activated carbon. Ethanol treatment reduced the lysing-algae activity. There were a large amount of ABC transporter, outer membrane protein, flagellin and a small amount of toxin in the key differential proteins under conventional and susceptible conditions. The results indicate that Vm 117-T6 does not dissolve algae through cell wall degradation, and the virulence effectors of Vm 117-T6 contain endotoxins, and the proteins related to the transport, secretion and cell adhesion may play an important role in the virulence of Vm 117-T6. This paper provides evidence for the analysis of the algae-lysing mechanism of this strain, and also lays a foundation for the prevention and control of this strain.

Abstract:As a major ginseng cultivator, China is among the top countries in the world in terms of total ginseng production each year. In order to diversify the use of ginseng resources, scholars have begun to study the pharmacological effects and active ingredients of ginseng stems and leaves. Studies have confirmed that probiotic fermentation of Chinese herbal medicine can significantly improve its efficacy, and the efficacy of fermented ginseng is better than feeding ginseng alone, and it has been concluded that ginseng can be fermented to improve its biological activity and enhance its efficacy after fermentation. However, not all microorganisms are suitable for fermentation of Chinese medicine, and the degree of suitability of microorganisms and Chinese medicine needs to be further explored. The application of fermented ginseng stems and leaves to aquatic animals has not been reported, and the research on the aptamer used for fermenting ginseng stems and leaves still needs to be studied. In order to study the effect of ginseng stem and leaf extract fermented by Lactobacillus casei on the immune and antioxidant function of Carassius auratus, the stem and leaf extract of ginseng was fermented with L. casei liquid of 3%, 4%, 5% and 6% (recorded as L3, L4, L5 ,L6), and then added to the basic feed (recorded as L0). The C. auratus with an initial average weight of (25.00±0.05) g was fed for five weeks. Periodic samples were collected from the serum, hepatopancreas, middle kidney, spleen and whole intestine of the C. auratus to detect the changes of relevant immune indexes. A protective test for Aeromonas hydrophila attack was performed at the end of the feeding trial. The results showed that alkaline phosphatase (AKP), immunoglobulin M (IgM), lysozyme (LZM), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were significantly higher in the group with L. casei seed inoculation of 5% (L5 group) compared with the control group, and malondialdehyde (MDA) activity was significantly lower in the L5 group than in the L0 group. The gene expression levels of IL-10, TNF-α, TGF-β, IFN-γ, and IL-1β were elevated to different degrees in all tissues, showing spatial and temporal differences, with the elevated gene level expression of IL-10 being more sensitive.In the protective attack test, the L5 group had a survival rate of 30%, the highest survival rate compared to the other groups.Under the present experimental conditions, when the inoculum of L. casei was 5%, the fermentation of ginseng stem and leaf extract was the most effective for the application to C. auratus , which could improve the antioxidant capacity and the expression of immune-related genes, and had a better preventive effect on reducing the infection of A. hydrophila. This experiment adheres to the concept of turning waste into treasure and green safety, which provides a theoretical basis for the application of probiotic fermentation of traditional Chinese medicine in practical production.

Abstract:To investigate whether grass carp (Ctenopharyngodon idella) TAB2 (CiTAB2) can interact with CiTAK1 and the effect of their interaction on the expression of two antimicrobial peptides (AMPs) genes Cihepcidin and Ciβ-defensin1, the temporal and spatial expression patterns of Citab2 and Citak1 in the immune-related tissues of grass carp after Vibrio mimicus infection were firstly analyzed by qPCR in the present study. Subsequently, fluorescence co-localization, co-immunoprecipitation and Western blot were used to identify the intracellular co-localization and interaction between CiTAB2 and CiTAK1 proteins. Finally, the mRNA expression levels of Cihepcidin and Ciβ-defensin1 were examined after the overexpression plasmids pEGFP-N1-Citak1 and pEGFP-N1-Citab2 were co-transfected into CIK cells. The results showed that V. mimicus infection significantly altered the relative mRNA expression levels of Citab2 and Citak1. Among them, the former showed different spatio-temporal expression patterns in each examined tissue at different time points post-infection, while the expression pattern of the latter was up-regulated then down-regulated in all examined tissues. CiTAB2 and CiTAK1 were co-localized in the cytoplasm of both HEK293T and CIK cells observed under a fluorescence microscope, and the CiTAB2-CiTAK1 protein complex could be formed in HEK293T cells post-transfection. After co-overexpression of CiTAB2 and CiTAK1, the mRNA expression levels of Cihepcidin and Ciβ-defensin1 were significantly up-regulated in the CIK cells at each test time point. These results indicated that CiTAB2 could interact with CiTAK1 and their interaction was able to promote the transcriptional expression of these two AMPs, which provides a new strategy for the control of fish vibriosis from the perspective of protein interaction.

Abstract:In order to establish an antiviral drug screening method targeting to Nrf2 / HO-1 pathway, the recombinant plasmids of Nrf2 and HO-1 promoter were constructed, respectively. The effects of different concentrations (0.0, 3.1, 6.3, 12.5, 25.0 and 50.0 μg/mL) of resveratrol, silybin, andrographolide and curcumin on promoter activity were detected by the dual luciferase reporter system in fathead minnow cells (FHM). Further, spring viremia of carp virus (SVCV) and rana grylio iridovirus (RGV) were used to verify the antiviral effect of positive drugs in FHM cells. The results showed that a variety of binding sites for transcription factors were found in the Nrf2 and HO-1 promoter sequences, including FOX family and IRF family. As well, 1 and 3 methylation-related CpG islands were found, respectively. The dual luciferase reporter assay showed that silybin, andrographolide and curcumin could activate the Nrf2 and HO-1 promoters. Drug concentration gradient experiments showed that curcumin and andrographolide at a concentration of 6.3 μg/mL could significantly up-regulate the promoter activities of Nrf2 and HO-1. The cytopathic effect observation, virus replication tests and virus titration assay confirmed that curcumin and andrographolide can inhibit the infection of SVCV and RGV significantly. In conclusion, the pGL3-Nrf2 and pGL3-HO-1 promoter reporter plasmids established in this experiment can be used for the screening of anti-aquatic virus drugs, and also provide tools for the study of the transcriptional regulation mechanism of Nrf2 and HO-1.

Abstract:Autophagy is an important process that maintains homeostasis in eukaryotic cells and is involved in cell differentiation, development, and immunity. However, little is known about the function of autophagy-related genes (ATGs) in the immune response of fish. To explore the function of ATG5 in virus infection of the Larimichthys croaker, we cloned a ATG5 gene from large yellow croaker (Larimichthys crocea) and its open reading frame (ORF) was 828 nucleotides long, encoding a protein of 275 amino acids. The predicted molecular weight of LcATG5 was 32.3 ku and its theoretical isoelectric point was 5.7. Homology comparison showed that LcATG5 had a highly conserved APG5 domain as found in other vertebrate ATG5 proteins. Phylogenetic analysis showed that LcATG5 was closely related to Collichthys lucidus ATG5. LcATG5 was detected in all eleven tissues of L. crocea, with highest expression in blood and lowest in spleen. LcATG5 transcripts were also detected in primary head kidney granulocytes, lymphocytes and macrophages, and L. crocea head kidney cell line (LYCK), with highest expression in primary head kidney granulocytes. After induction by poly(I:C), LcATG5 transcripts were significantly increased in these cells, and were more responsive in LYCK cells, with 3.93-fold increase at 12 h. In the LcATG5-overexpressed epithelioma papulosum cyprini (EPC) cells, the cytopathic effects (CPE) caused by spring viremia of carp virus (SVCV) infection were increased at 48 h post-infection. The viral titer of SVCV in the culture supernatant of the LcATG5-overexpressed EPC cells was 10^13.82 TCID₅₀/mL, which was higher than that of control group (10^9.27 TCID₅₀/mL). Besides, the expression levels of three viral genes (SVCV-G, SVCV-M, and SVCV-P) were significantly up-regulated in the LcATG5-overexpressed cells with 13.77-, 15.72-, and 11.39-fold increases, respectively. These results indicate that LcATG5 plays a role in promoting virus replication, which may provide a basis for in-depth study of the function and mechanism of autophagy and ATGs during virus infection in fish.

Abstract:The second national pollution sources survey showed that the total nitrogen emission from aquaculture is 99100 tons in 2017. To protect the environment and human health, it is important to remove nitrogen from aquaculture wastewater before being discharged to surrounding waters. Biological denitrification is considered the most promising approach methods, since nitrate can be reduced to harmless nitrogen gas by bacteria. Sufficient carbon source is necessary during heterotrophic denitrification process. To solve the problems mentioned above, external carbon sources such as methanol, acetic acid and glucose were added to the wastewater, whereas they were generally high-cost, high-energy and high operating requirement. In contrast, agricultural wastes were used as carbon source, which has shown significant economic advantages and high-efficiency. Many aquaculture wastewater treatment systems often face variations in hydraulic retention time (HRT) and Influent nitrate concentration (INC) which are caused by acute change of wastewater characteristics and production, and HRT and INC often exert a profound effect on the treatment performance of biological treatment systems. The purpose of this study is to construct a solid-phase denitrification system with loofah sponge as carbon source, and investigate the effects of HRT and INC on the denitrification performance of loofah sponge-denitrification reactor (LS-DR), so as to provide a theoretical basis for the further optimization of denitrification process of loofah sponge as denitrification carbon source in aquaculture tailwater. Loofah sponge, one typical agricultural waste, was studied as the carbon source for solid phase denitrification under dynamic flow conditions by using 1-D column experiment. We aim to preliminarily investigate the LS-DR’s NO₃^--N, NO₂^--N, NH₄⁺-N, TN, TP and COD removal effect at different HRT (16, 20, 24 and 28 h) and INC (50, 75, 100 and 125 mg/L). The optimal HRT of denitrification reactor was optimized by one-way ANOVA analysis. And, the high-throughput sequencing technology based on Illumina MiSeq platform was used to analyze the bacterial community structure of LS-DR in the initial and final stages of operation. The results indicated that when INC=50 mg/L and HRT=24 h, the removal efficiency of both NO₃^--N and TN in LS-DR reached the highest value, which were 98.97%±0.52% and 97.84%±0.94% respectively. And NO₂^--N was also at a low level (< 0.5 mg/L). On the basis of HRT of 24 h, when INC increases to 75, 100 and 125 mg/L, the nitrate removal efficiency and nitrate removal rate (NRR) of LS-DR increased significantly with the increase of INC (P< 0.05), and the effluent COD decreased with the increase of INC, but LS-DR did not realize complete denitrification. It is worth noting that LS-DR can completely remove NH₄⁺-N throughout the experiment. After 14 days of operation, SEM results showed that the surface structure of LS was conducive to the attachment and growth of microorganisms; high throughput sequencing results showed that the dominant bacteria of LS-DR included Proteobacteria, Bacteroidetes, Campilobacterota, Firmicutes and Verrucomicrobiota. Among the identified bacteria, Thermomonas (1.46%), Thauera (0.55%), Azospira (3.32%), Simplicispira (1.01%), Pseudoxanthomonas (0.39%), Herbaspirillum (3.02%) and Uliginosibacterium (0.9%) can carry out denitrification. Cyphaga xylanolytica (1.61%) and Cloacibacterium (2.69%) are mainly involved in the degradation of towel gourd, Flavobacterium (1.17%) and Diaphorobacter (0.64%) can both denitrify and degrade LS. According to the analysis of the above results, it is considered that the optimal HRT of LS-DR is 24 h and the optimal INC is 50 mg/L. This study provides a reference for the optimization of loofah sponge solid-phase denitrification process and promotes the development and application of new slow-release carbon sources.

Abstract:Isinglass is one of the traditional fish products in China, with unique flavor and taste. However, shortcomings such as soft taste and loss of nutrients after sterilization, limit its commercial development. In order to improve the heat sterilization process and reduce the deterioration of the quality of Sciaenops ocellatus isinglass after sterilization, the corresponding sterilization process at different sterilization temperature with F=4.5 min was investigated. Through the analysis of physical property, low-field nuclear magnetic and infrared chromatography, the effects of different sterilization temperature on the texture, color, collagen content, water distribution and protein structure of isinglass were investigated. The results showed that the isinglass products needed to be sterilized for 56.44, 15.85, 3.46 and 0.92 min at 110, 115, 121 and 125 ℃, respectively, to reach the F=4.5 min, and corresponding products could reach the commercial sterile state. In the range of 110 to 125 ℃, the brightness L^* value and b^* value decreased gradually from 59.78 to 49.65 and 17.80 to 11.25, while a^* value didn’t change notably (P>0.05), with the increase of the sterilization temperature. In addition, FTIR confirmed the triple helical structure of the collagens. The significant improvement of hardness and shear force of isinglass and the reduction of viscosity were also observed (P<0.05). After being sterilized at 125 ℃, hardness and shear force values were 37.02 g and 34.2 g·s, respectively, the viscosity was –2.82 g. Meanwhile, the collagen content of the sample rose from 7.60% to 14.92% and the collagen concentration in the solution declined from 3.33 mg/mL to 0.15 mg/mL. After being sterilized at 110 ℃, the degree of freedom of water in the isinglass was the highest, the proportion of free water in the isinglass gradually decreased with the increase of the sterilization temperature. Therefore, under the sterilization condition of higher temperature, the brightness of the isinglass was reduced, but the quality was improved with better texture and higher content of collagen. This study has important practical significance for promoting the processing and utilization of isinglass and improving the quality of sterilized ready-to-eat aquatic products.

Abstract:Enhancement and releasing are significant for the restoration of Sepiella japonica. To increase the survival rate of S. japonica dring transportation of proliferation and releasing, the effects of different oscillation frequencies (0, 60, 100, 120, 140 and 160 r/min) on death rate, the contents of lactate and glycogen, as well as enzyme activity of adult S. japonica were studied. The results showed that the death rate increased with the frequency increases in other groups, except for a slight decrease at the frequency of 160 r/min. The highest death rate was 97.1% at the frequencies of 120 and 160 r/min, and it was obviously higher than that at the frequencies of 0, 60 and 100 r/min (P<0.05). The death time mainly range from 4-8 h after oscillation and the lower the frequency, the later death time appeared. Except for some statistics fluctuations in a few groups, it basically followed the trend that glycogen content in the two tissues decreased with the increase of frequency, while the lactate content increased. Compared to the treatment group, the experimental group at the frequency of 140 r/min had a significant difference in lactate and glycogen contents(P<0.05). Except for the experimental group at the frequency of 60 r/min, the activity of alanine transaminase (ALT) and glutamic-oxalacetic transaminase (GOT) in pancreas tissues decreased, and the activity of superoxide dismutase (SOD) increased with the increase of frequency. Compared to other experimental groups, these three enzymes at the groups of 120, 140 and 160 r/min respectively, had a significant difference (P<0.05). The activity of alkaline phosphatase (ALP) increased with the increase of frequency, the groups of 140 r/min and 160 r/min had a significant difference compared with other groups (P<0.05). The death rate of squid was positively correlated with SOD, lactate content and ALP, and negatively correlated with muscle glycogen content, GOT, ALT and digestive gland glycogen content (P<0.01). In conclusion, during the transport of S. japonica, energy exhaustion and hepatopancreas damage could be the major reasons of high death rate. In order to solve this problem, the oscillation frequency during transportation should be below 100 r/min and the transportation time under 3 hours and should avoid strong waggle.