Abstract:Aquaculture of abalone has become an important part of China's marine economic development. In recent years, mass mortality events of farmed abalone caused by hypoxia stress, that combined with hypoxia, had often occurred, which had brought serious economic losses to the abalone aquaculture industry and had become an important environmental factor hindering the healthy and sustainable development of the abalone aquaculture industry. In this paper, research progress on the mechanism of response to hypoxia in abalone is reviewed from the perspectives of growth, survival, physiological and biochemical indexes (including, heart rate, metabolism, enzyme activity, pH, etc.), immune function as well as stress response genes and their expression regulation, with a view to providing a reference for further research on abalone's mechanism of response to hypoxia stress and developing new varieties of abalone resistant to hypoxia. In addition, the prevention and control measures of abalone hypoxia stress are also proposed, hoping to help farmers reduce the losses caused by the large-scale death of cultured abalone-caused hypoxic stress in summer.

Abstract:Medaka (Oryzias latipes) is one of the model organisms for studying gene function, organogenesis, and development mechanism. Although editing technology has been widely used in medaka, there was no detailed report on the use of CRISPR/Cas9 to establish O. latipes mutant. To thoroughly describe the establishment process of O. latipes mutant, this study takes the knockout of Olpax6.1 as an example. Firstly, the gRNA target of Olpax 6.1 gene was designed using the ChopChop website; secondly, the template for in vitro transcription of gRNA and Cas9 mRNA were obtained by PCR and plasmid digestion respectively, and gRNA and Cas9 mRNA were synthesized by in vitro transcription; thirdly, the mixture of Cas9 mRNA and gRNA was microinjected into medaka embryos to obtain the mutated F₀; finally, we used PCR, T7 Endonuclease I digestion and Sanger sequencing to screen the offspring of the cross between F₀ and wild type to obtain F₁ with the same mutation type, and then crossed the same mutated F₁ and screened their offspring to obtain Olpax6.1 knockout homozygous mutant, which displays the classic phenotype of ocular developmental malformations. Taken together, this study provides technical guidance for the generation of mutants in O. latipes and other fish.

Abstract:Dissolved oxygen is an important environmental factor affecting the survival of fish. In this study, the value of dissolved oxygen at critical oxygen partial pressure (Pcrit) and loss of equilibrium (LOE), respiratory rate, plasma cortisol, glucose, white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), hematocrit (HCT) and the alternation of gill morphology and related parameters [lamellar length (SLL) and width (SLW), interlamellar distance (ID) and perimeter] as well as the proportion of secondary lamellae available for gas exchange (PAGE) were observed during hypoxia to illustrate hypoxia tolerance, physio-biological response and morphological changes in the gills of Sebastes schlegelii during hypoxia stress. Results showed that the value of dissolved oxygen at Pcrit and LOE of S. schlegelii at (88.01±5.34) g were (3.96±0.11) mg/L and (2.60±0.21) mg/L respectively under the condition of water temperature (15.6±0.2) ℃, pH value 7.85, and salinity 30.0. The PAGE and respiratory frequency increased first and then decreased, the highest value respectively obtained at Pcrit and LOE (P<0.05) throughout hypoxia stress. Meanwhile, plasma cortisol and glucose levels significantly increased, the highest values observed at LOE and Pcrit (P<0.05). In blood physiology, Hb, HCT, WBC similar results like plasma cortisol, with the highest value at LOE (P<0.05), whereas RBC remained unchanged during hypoxia stress. Besides, the SLL, ID, Perimeter of gills increased significantly during hypoxia stress, and reached the maximum value at LOE point (P<0.05) either; the SLW decreased and reached the minimum value at the LOE (P<0.05). At the same time, the clubbing, hypertrophy, hyperplasia and epithelial cell edema were observed in the secondary lamella. All the aforementioned parameters recovered to normal levels after re-oxygenation 24 h in S. schlegelii. In conclusion, S. schlegelii are sensitive to low dissolved oxygen, have numbers of changes in hematology and morphology for hypoxia stress and reduce hypoxia stress after reoxygenation treatment for 24 h. These findings expand current knowledge on hypoxia tolerance and help in the management of S. schlegeli in captivity.

Abstract:Beak is one of the important hard tissues of Cephalopod, which is widely used for age and growth estimation, population structure analusis and resource assessment of Cephalopod. The microstructure and the morphological growth characteristics of beak of Sthenoteuthis oualaniensis were studied with morphometric parameters of 260 pairs of beaks from 1009 samples collected in Northwest Indian Ocean by a Chinese lighted cover net fishing vessel during survey from March to May 2019 in combination with their ages. The result indicated that there were obvious growth increments in the upper beak rostrum sagittal section (RSS), and increments in hood region were more clear. The analysis of covariance indicated that there was no significant difference between sex and age in the morphological growth characteristics parameters and relative size of beaks. The results of Akakchi Information Criterion (AIC) showed that the linear functions were found to be the optimal for the relationships between all the morphological characteristic parameters and age except UHL, and the logarithmic function was the optimal for UHL growth. The relative size decreased with the increase of age, and the variation trend was basically the same. The absolute and relative growth rates of the six morphological characteristic parameters showed a decreasing trend, and the absolute growth rate of ULWL and LWL reached the peak at 181-210 d but the absolute growth rates of UCL、UHL、LLWL and LCL reached to the peak at 211-240 d, while the instantaneous relative growth rates of all the parameters peaked at 181-210 d. This study laid a foundation for further study on the age and growth, population structure and life history of S. oualaniensis based on the microstructure of the beak, and provided a scientific basis for scientific exploitation of this resource.

Abstract:Artificial reef is an effective measure for improving benthic habitat, macrobenthos diversity, and richness. Its ecological effects are closely related to the duration of placement. In order to explore the effect of reef construction duration on the trophic relationship of macrobenthos, this study applied stable isotope techniques to compare the carbon and nitrogen stable isotope compositions of macrobenthos and their food sources in artificial reef areas of different ages and control areas in the coastal waters of Furong Island, Laizhou. In addition, the food bases and trophic levels of macrobenthos in different areas were also calculated. The results showed that: ① The δ¹³C values of macrobenthos in long-age reef area, short-age reef area and control area ranged from −25.08‰-−13.34‰, −25.86‰-−17.80‰, and −25.39‰-−11.06‰, respectively. The corresponded δ¹⁵N values ranged from 10.73‰-15.78‰, 10.02‰-14.89‰, and 10.17‰-15.80‰, respectively. ② Compared to short-age reef and control area, the macrobenthos community in the long-age reef area has higher trophic diversity, higher diversity of food sources, more diverse trophic niches within the community and better stability of community structure. ③ According to feeding habits, macrobenthos can be divided into four functional groups, namely planktonophagous group (Pl), carnivorous group (C), detritivorous group (D) and omnivorous group (O). Among the four potential food sources (phytoplankton, zooplankton, particulate organic matter (POM) and sediment organic matter (SOM)), phytoplankton had the highest average contribution (31.40%) to the three types of consumers, except for carnivorous group. ④ The trophic levels of consumers in long-age reef , short-age reef , and control area were 2.00–3.83, 2.00–3.49 and 2.00–3.87, respectively. In addition, higher trophic level predators were observed in long-age reef area, implying more complex trophic structure in long-age reef area than that of other areas. The results of this study showed that the construction of artificial reefs could enhance the utilization of carbon sources of phytoplankton by macrobenthos, increase the trophic diversity and richness of macrobenthos community, and increase the numbers of higher trophic animals in the community. The results of this study contribute to the existing knowledge for understanding the ecological effects of artificial reef construction and provide basic information for further studies on the material cycle and energy flow of benthic food web.

Abstract:In order to explore the impact of different substrate types after the deployment of artificial reefs on the community structure characteristics of benthic swimming species, six cruises of field surveys were conducted in the artificial reef area located in the Xiaozhushan Island. The survey area was divided into three transects with a radius of 200 m, 400 m, and 600 m based on the artificial reef modules deployed in the center of the sea area. Trap and visual census methods were combined to sample benthic swimming organisms in June, August 2017, June 2018 (twice), and June, July 2019. Relative importance index (IRI), principal coordinate analysis (PCoA) and one-way analysis of variance (ANOVA) were utilized to explore the variance of spatial-temporal constitution of the community, and General additive model (GAM) was utilized analyze the effects of environmental factors to catch per unit effort (CPUE) and biodiversity index of the community. The results showed that two sampling methods totally obtained 47 species in 5 categories, including 38 species from trap, 28 species from visual census and 19 species in common. IRI and PCoA indicated that Charybdis japonica, Asterina pectinifera, Hexagrammos otakii, Sebastes schlegelii, and other fusiform fish were mainly captured by trap; Echinodermata such as A. pectinifera, Palaemon graviera, Tridentiger trigonocephalus, Amblychaeturichthys hexanema, Triaenopogon barbatus and Chaeturichthys stigmatias were mainly captured by visual census. One-way ANOVA of both sampling methods indicated that the CPUE of 200 m and 400 m transects was significantly higher than 600 m transect, but there was no significant difference between years. GAM showed CPUE increased with the rising complexity of substrate types and duration of the deployment of artificial reefs, was positively correlated with substrates of artificial reef and rock, and was affected by salinity and temperature. In addition, GAM showed that biodiversity index of trap was significantly higher than visual census, and it was influenced by salinity and transparency. But there was no connection with substate types. This study revealed the effects of different substrate types to benthic swimming organisms, proved the complementary of the combination of trap and visual census, and provided a reference for the management of reef substrate.

Abstract:Anhui is one of the main areas of artificial crawfish (Procambarus clarkia) farming in China. To understand the genetic diversity of cultured P. clarkii populations in Anhui Province, this study selected three artificial cultured populations of P. clarkii in Anhui (WH, XC and HF) as the research object, with two wild population of P. clarkii in Anhui (TL and MAS) and four artificial cultured populations (JL, JH, GH and XH) as control. Ten pairs of P. clarkii microsatellite primers were selected to study microsatellite genetic diversity and genetic structure. The results revealed that the genetic diversity of P. clarkii artificial cultured in Anhui was higher than wild population and four artificial cultured populations (JL, JH, GH and XH), and the XC population had the highest genetic diversity. Tests of deviation from Hardy-Weinberg equilibrium indicated all 10 loci in 9 populations were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and most of them had significant heterozygosity deficiency．AMOVA analysis showed that genetic variation was determined within the population. F_st in most groups showed moderate differentiation (0.05 < F_st < 0.15). The gene flow demonstrated there were extensive gene exchanges between different populations, particularly between GH and JH. UPGMA tree revealed that XC and HF belonged to the same clade, and WU、GH、MAS and JH were in the other clade, whereas the remaining populations formed their own clade. STRUCTUR results showed that most individuals in XC and HF populations were assigned to the same genetic population, suggesting that they may have the same origin. The results showed that the cultured population of P. clarkii in Anhui had high genetic diversity, which provided reference for the protection and improvement of P. clarkii germplasm resources in Anhui.

Abstract:Bioturbation refers to the biological reworking of soils and sediments by benthic animals, especially macrobenthos (e.g Venerupis philippinarum, Nutaliaolivacea, and Echinocardium cordatum, etc.). Previous studies have shown that bioturbation activity could enhance the migration and transformation of inorganic nitrogen with different forms at the sediments-water interface on the tidal flat, and increase the benthic nitrogen release rate, thus affecting the chemical characteristics of the sediment. In this study, Urechis unicinctus was selected as the research object to explore their effect of bioturbation on the diffusion flux of nitrogen and phosphorus at the sediment-water interface. Chinese Penis Fish, U. unicinctus, is an echiuran species with solid environmental adaptability, and is widely distributed in North Korea, Japan, and northern China, especially near intertidal and subtidal zones along the Bohai Sea coast of China. Usually, U. unicinctus inhabit in U-shaped burrows in sediments in intertidal areas and feed on filtering organic particles in the water. Furthermore, it has high economic value and nutritional value as aquatic products. Currently, the research of U. unicinctus mainly focuses on Reproductive biology, tolerating mechanism for sulfide, extract the active component, ecological health cultivation, etc. However, very few studies can be found about the impact of U. unicinctus bioturbation on the biogeochemical process of biogenic elements. To understand the ecological role of U. unicinctus in the sedimental environment, the effect of biological disturbance of U. unicinctus on the diffusion of nitrogen and phosphorus at the sediment-water interface was studied with indoor experiment. The U. unicinctus were divided according to four areal densities: low density (LD, 500 ind/m²), medium density (MD, 2 500 ind/m²), high density (HD, 8 300 ind/m²) and control group (CO, no worms), respectively. Each of this treatment group has five replicates. The 20-day indoor experiment were carried out in tanks during November-December 2020.The results showed that the release rate of NH₄⁺ contents from sediment to water ranged from 10.6 to 765.3 μmol/(m²·d), and it increased to the maximum and then decreased gradually with time elapsed. Compared to the control group, the average nutrient release rate increased by 39%, 111%, and 257% in the LD, MD, and HD treatment groups, respectively. Significant differences were found for NH₄⁺flux between the LD, MD, HD groups and the CO group (P<0.05). The values of NO₃^-+NO₂^-flux ranged from −172.05 to 208.63 μmol/(m².d) for all the experiment, but the average flux values were reduced by 8% and 32%, and 81%, respectively, in the LD, MD, and HD treatment groups. Further, the values of PO₄^- flux ranged from −7.85 to 6.42 μmol/(m².d) in the treatment groups during the entire period. There was a significant difference in PO₄^--P flux between the MD and HD groups and the CO group from day 2 to day 14 during the experiment (P<0.05). It turned out that NH₄⁺ is the main dissolved inorganic nitrogen (DIN) that affected by the biological disturbance of U. unicinctus. Taking into account the influence of U. unicinctus's metabolism and ammonia excretion, the NH₄⁺ diffusion flux is calculated by subtracting the ammonia excretion. Although the NH₄⁺ diffusion flux has a slight decrease, the trend of change over time remains unchanged. The results indicated that although the ammonia excretion effect of U. unicinctus has a certain contribution to the diffusion of NH₄⁺ from the sediment to the water body, the dominant factor still comes from the bioturbation effect of U. unicinctus. In addition, the U. unicinctus promoted the diffusion of DIN from sediments to bottom water, with an obvious density effect. This work provides support to evaluate the potential ecological role of the U. unicinctus in the integrated multi-trophic aquaculture (IMTA) system.

Abstract:Grouper is a rare seawater fish with delicious taste and rich nutrition. In our previous study, we bred the hybrid “Jinhu grouper” with a growth advantage by crossing the frozen sperm of Epinephelus tukula and E. fuscoguttatus egg. The average weight of 1 age old “Jinhu grouper” was 559.76 g, 1.63 times that of the hybrid of E. fuscoguttatus♀×E. lanceolatus♂. In this study, the compositions and contents of water, protein, crude fat, ash content, amino acids, fatty acids and 10 mineral elements in the muscles of E. fuscoguttatus, E. fuscoguttatus♀×E. tukula♂ and E. fuscoguttatus♀×E. lanceolatus♂ were measured by conventional methods, according to the national standard. The crude protein contents in the muscles of E. fuscoguttatus, E. fuscoguttatus♀×E. tukula♂ and E. fuscoguttatus♀×E. lanceolatus♂ were 19.0%, 20.2% and 16.8% respectively. The total quantity of amino acids, essential amino acids, essential amino acid index and the fresh amino acids of these three groupers were (18.10%, 18.53%, 16.01%)，(7.52%, 7.70%, 6.32%)，(88.20, 84.80, 88.30) and (6.92%, 7.13%, 6.10%), respectively. The fat content in the muscle of three grouper was 3.0%, 4.2% and 2.4%, respectively; and the content of DHA+EPA in E. fuscoguttatus♀×E. lanceolatus♂ was 24.02%, the content of DHA+EPA in E. fuscoguttatus♀×E. tukula♂ was 23.56%, the content of DHA+EPA in E. fuscoguttatus was 22.76%. There are abundant mineral elements in the muscles of three grouper; five major elements (K, Ca, Na, Mg and P) and five trace elements (Fe, Mn, Cu, Zn and Se) were detected. The research shows that E. fuscoguttatus, E. fuscoguttatus♀×E. tukula♂ and E. fuscoguttatus♀×E. lanceolatus♂ can provide abundant protein, fat, inorganic salt and mineral elements for humans, thus the three groupers are excellent mariculture varieties with high edible value. Moreover, some nutritional indexes of the hybrid E. fuscoguttatus♀×E. tukula♂ was significantly higher than the female parent E. fuscoguttatus.

Abstract:Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in China, with annual culture production of around 800, 000 tons. Although more than 90% of adult E. sinensis have green carapace and limbs, there are a very small percentage of crabs with uncommon carapace color in wild or domesticated populations, including purple, yellow, red and white ones. Color is an important economic trait for customer acceptance and economic value of Chinese mitten crab, making it a potential target trait for the genetic breeding of this species. E. sinensis with white carapace is a new strain introduced by genetic breeding programs, which has several characteristics including heritable white color and fast growth rate. However, it remains unclear how the white strain compares to the normal green strain of adult E. sinensis in total edible yield and nutritional quality. Therefore, juvenile crabs of the two strains were reared in the same pond until the commercial size, and their edible yield, carotenoid content and nutritional composition were compared. The study showed the following. ① There were no significant differences in tissue coefficient, edible yield, and conditional factor between white and green E. sinensis. (P>0.05). ② The lightness (L^*) value in wet and dry samples of the white strain was significantly higher than that of the green strain, but the redness (a ^*) value was significantly lower in the white strain (P>0.05). No significant differences were found in color parameters in hepatopancreas and ovaries (P>0.05). ③ The contents of total carotenoid and astaxanthin in the carapace of the white strain were significantly lower than those of the green strain (P<0.05), but there were no significant differences in hepatopancreas and ovaries between the two strains (P>0.05). ④ There was no significant difference in the content of proximate composition in edible tissue while some differences existed in fatty acid composition between the two strains. The C20:4n6 in the muscle of white females was significantly higher than that of green strain (P<0.05). However, 18:0, 18:2n6 and docosahexaenoic acid (DHA) / eicosapentaenoic acid (EPA) ratios in hepatopancreas of green females were significantly higher than those of white females (P<0.05). ⑤ Irrespective of sex, there were no significant differences in the composition of most amino acids and essential amino acid scores between the two strains (P>0.05), and white females had significantly lower levels of isoleucine and tyrosine in muscle than green females (P<0.05). In conclusion, although the white carapace strain of E. sinensis had the lower color parameter and carotenoids content in the carapace than the green strain, the two strains had similar level of edible yields, proximate composition, the contents of major carotenoids, fatty acid composition and amino acid contents in their edible tissues, which suggested the white carapace strain of adult E. sinensis has similar nutritional quality with green strain and potential value for the farming industry of this species.

Abstract:An 8-week feeding experiment was conducted to investigate the effects of dietary n-3/n-6 fatty acid ratios on the growth performance, muscle quality, antioxidant capacity, serum physiological indicators and digestibility of Macrobrachium rosenbergii. Five iso-nitrogenous and iso-lipidic diets were formulated with graded levels of n-3/n -6 PUFA(D1: 0.37, D2: 0.59, D3: 0.93, D4: 1.51, D5: 4.38), with four replicates, 40 shrimps each. The results showed that the dietary n-3 PUFA/n-6 PUFA ratio did not significantly affect survival rate (SR). The final weight (FW)，weight gain rate (WGR) and specific growth rate (SGR) of the shrimp first increased and then declined with the increase of dietary n-3 PUFA/n-6 PUFA ratio, with the D3 group displaying the highest values. The D3 group also displayed the highest hepatopancreas protease and fatty acid synthetase (FAS) activities. Dietary n-3 PUFA/n-6 PUFA ratio had no significant effect on the muscular crude protein content, moisture and ash content of the shrimp, but the D3 group showed the highest muscular total lipid content which was significantly higher than those of other groups. The variation trend of muscular n-3 PUFA/n-6 PUFA ratio in shrimp was positively correlated with the variation trend of dietary n-3 PUFA/n-6 PUFA ratio. The superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) in hepatopancreas and serum as well as the ceruloplasmin (CP) content in serum first increased and then decreased with the increase of dietary n-3 PUFA/n-6 PUFA ratio and peaked in D3 (0.93) group and D4 (1.51) group, but the content of malondialdehyde (MDA) rose continuously. The content of total cholesterol and triglycerides in the serum of D1 group were significantly higher than those of other group as the dietary n-3 PUFA/n-6 PUFA ratio increased. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of the shrimp first declined and then increased and the D3 group had the lowest values. It is therefore suggested that an appropriate dietary n-3 PUFA/n-6 PUFA ratio significantly improves the growth performance and antioxidant capacity the juvenile prawn M. rosenbergii. Based on Broken-line regression analysis of WGR and SGR, the optimal dietary n-3 PUFA/n-6 PUFA ratio, for growth performance of juvenile freshwater giant prawn M. rosenbergii is 0.86-0.94.

Abstract:In the last decades, the marine cage-cultured large yellow croaker (Larimichthys crocea) in Fujian province suffered from Megalocytivirus frequently. The Megalocytivirus isolated from L. crocea have not found any sensitive cell-culture model yet. The missing of culture methods has set back the studies on Megalocytivirus from L. crocea. The Siniperca chuatsi cell line mandarin fish fry cell line-1 (MFF-1) derived from S. chuasti fry, which has been proved to be highly sensitive to multiple ISKNV-like and RSIV-like Megalocytivirus members, may also be a promising culture system for L. crocea Megalocytivirus. To establish methods for cell culture and classification of Megalocytivirus strains from L. crocea, and to make a comparison of their major capsid protein (mcp) genes, which will benefit for the studies of invasion and prevention of these unclassified Megalocytivirus, kidney tissue homogenates of Megalocytivirus-positive L. crocea juveniles (FD201807 and SA201808) were inoculated to the MFF-1 cell line and subcultured continuously. From the tissue homogenates and freeze-thawed infected cells, the virus genome was extracted. The virus mcp was then cloned and sequenced, and compared with the NCBI GenBank records of Megalocytivirus, and a 2018–2020 Fujian collection of 15 Megalocytivirus isolates from L. crocea as well. The results showed both two Megalocytivirus isolates caused typical cytopathic effects (CPE) on MFF-1 cells after 3 passages, of which the key features included cell rounding and shrinking, increased cell diopter, continuous cell detachment and particulates secretion with time. Hexagonal viral particles and empty capsids with a size of 130-150 nm were observed in the cytoplasm of infected MFF-1 cells under a transmission electron microscope (TEM). With the processing of virus subculture, the CPE interval of FD201807 shortened from 10 d to 3-5 d, while which of SA201808 remained 7-8 d. mcp gene revealed a 21-bases difference between SA201808 and FD201807. Phylogenetic and clustering analysis indicated that the mcp gene of SA201808 was highly homologous to LYCIV-Zhoushan (GenBank: MW139932. 1), while the homological identity of mcp gene between FD201807 and lateolabrax maculatus iridovirus (LMIV, GenBank: MH577517. 1) was up to 99. 93%. 12 of the total 15 Fujian L. crocea Megalocytivirus isolates were found clustered with SA201808, and the other 3 were clustered with FD201807. In this study, L. crocea Megalocytivirus were isolated through MFF-1 cell culture, indicating the differences among L. crocea Megalocytivirus strains, which could benefit for better understanding of this virus group.

Abstract:Grass carp (Ctenopharyngodon idella) is one of the most important aquaculture fish species in China. C. idella hemorrhagic disease caused by grass carp reovirus (GCRV) results in severe outbreaks annually. It causes great losses to aquaculture industry and a threat to food safety. GCRV is the most virulent in aquareovirus. VP56 is a fibrin which is specific to type Ⅱ/Ⅲ. It attaches the surface of host cells during GCRV infection. In order to clarify the infection mechanism of GCRV, this study delved into the interaction between VP56 and host cell proteins. First, co-immunoprecipitation (co-IP) was performed. The specific antibodies to different tags respectively fused on the two molecules were used. Empty vectors and negative immunoglobulin G were used as control. It was found that VP56 interacts with the molecular chaperone protein glucose regulated protein 78 ku (GRP78) located in the endoplasmic reticulum. Then, the interaction between VP56 and GRP78 was verified by the bimolecular fluorescence complementation (BiFC) based on the far-red fluorescent protein mNeptune. According to the BiFC results, red fluorescence of mNeptune was observed in CIK cells transfected with both VP56 and GRP78 respectively linked with each terminal of the mNeptune protein, showing that these two fusion proteins presented closely and sent out red fluorescence signal. In VP56 stably expressed C. idella kidney (CIK) cells, the endoplasmic reticulum morphology underwent tremendous changes, compared with empty vector stably expressed CIK cells resulting in swelling, expansion, and degranulation, indicating that VP56 activates endoplasmic reticulum stress. mRNA levels of transcription factors downstream of endoplasmic reticulum stress were detected, which revealed that VP56 activates the signal transduction pathway regulated by activating transcription factor 6 (ATF6) and triggers the unfolded protein response. The above results indicate that during GCRV-Ⅱ infection, cell homeostasis is destroyed, and endoplasmic reticulum stress is activated. This study provides new ideas for the study of GCRV infection mechanism and anti-GCRV research, reveals a new virus escape strategy, provides in-depth antiviral research in C. idella, and contributes to the prevention and control of C. idella hemorrhagic disease in the freshwater aquaculture industry.

Abstract:In recent years, an epidemic disease called "zombie disease" by local farmers has appeared in the ridgetail white prawn (Exopalaemon carinicauda) breeding area of Jiangsu Province. The main symptoms of the disease include reddish body color, whitish muscles and milky liquid under the carapace. The diseased prawns usually show slow movement and reduce food intake and died quickly after leaving the water. In order to determine the pathogen and epidemiological characteristics of the "zombie disease"in farmed E. carinicauda in the coastal area of Jiangsu Province since the winter of 2018, LB medium and PDA medium were used to isolate colonies with a diameter of 1-3 mm, neat edges, and beige-colored elevation from the hemolymph of diseased shrimp, which was named MQ2101. This strain was identified as Metschnikowia bicuspidata by means of morphologic structure observation, 18S rRNA gene analysis and constructing phylogenetic tree. Furthermore, the pathogenicity of the strain MQ2101 was confirmed by the infection experiment in healthy E. carinicauda. The results showed the infected E. carinicauda died with the similar symptoms to naturally morbid E. carinicauda and the same bacterium could be isolated from the infected E. carinicauda. The LD₅₀ of strain MQ2101 to E. carinicauda was 1.39×10⁷ CFU/ind. Histopathology and transmission electron microscopic observation showed that there were different degrees of lesions in gills, muscle and hepatopancreas, of which hepatopancreas lesions were the most serious, with hepatic tubules vacuolated and lumen volume enlarged. Moreover, a large number of colonized bacteria were found in gill and hepatopancreas. Epidemiological findings showed the disease frequently occured in the cool season (February to May), mainly erupted in prawn and crab mixed ponds, with a incidence rate of 5%-30% and a mortality rate of 3%-10%, the morbidity was increasing year by year. In the present study, M. bicuspidata was identified as the pathogen of "zombie disease" in E. carinicauda in coastal areas of Jiangsu Province, which is highly pathogenic to E. carinicauda and mainly infects the hepatopancreas and gills. It was the first time that revealed M. bicuspidata infection in E. carinicauda and the pathological changes caused by the infection. The above findings provide a scientific basis for the prevention and control of "zombie disease" in the culture of prawns.

Abstract:Eriocheir sinensis is one of the most important aquaculture species in China. With intensification of high-intensity feeding and degradation of the environment, the increasing diseases caused by bacteria infections have resulted in enormous economic loss in crab aquaculture. Akt is a core molecule of PI3K-AKT signaling pathway, which is highly conserved from invertebrate to vertebrate that plays essential roles in various physiological and pathological processes, such as cell proliferation, metabolism, development and survival. Although several Akts among invertebrates have been explored, the identities of Akt and their downstream effectors, especially in pathogen infection of crustaceans, remains poorly understood. In the present study, an Akt gene (designated as EsAkt) was identified in Chinese mitten crab E. sinensis, and the mRNA expression patterns of EsAkt in response to immune stimulations were investigated. The specific primers and PCR technology were used to clone and obtain the E. sinensis Akt gene (EsAkt). The expression level EsAkt in different tissues and after different immune stimulations were detected by real-time quantitative PCR (qRT-PCR). The cellular localization of EsAkt protein was detected by immunofluorescence assay. The changes of apoptotic gene after EsAkt RNA interference were analyzed by double stranded RNA interference. The results showed that EsAkt molecule contained the PH domain, the central catalytic domain (S_TKc) and the carboxy-terminal hydrophobic domain (S_TK_X). EsAkt was constitutively expressed in all tested tissues, and its expression in immunity related tissues (such as hepatopancreas, gills and blood lymphocytes) was significantly higher than that in stomach and intestine (P<0.01). Immunofluorescence assay results indicated that the oval hemocytes were observed in the bright field, and the cell nuclei were stained by DAPI in blue. The EsAkt-reactive areas were stained in green, and the positive green signals were mainly diffuse localization in the cytoplasm. After injecting lipopolysaccharide (LPS) and Aeromonas hydrophila into E. sinensis, the expression of EsAkt was significantly induced. After LPS immune stimulation for 12 h, the response of EsAkt reached the peak, which was 4.35 times that of the control group (P<0.01). After stimulation for 48 h, the mRNA expression level of EsAkt decreased, but it was still 2.62 times that of the control group (P<0.01); After 6 h of A. hydrophila immune stimulation, the response of EsAkt reached the peak, which was 9.05 times higher than that of the control group (P<0.01). After 48 h of stimulation, the mRNA expression level of EsAkt fell back to normal. Upon double stranded RNA interference with EsAkt, the mRNA expression level of EsAkt was 0.38 times higher than that of the control group (P<0.05), while the expression level of apoptosis related gene Escaspase-3-like was significantly up-regulated, which was 2.69 times higher than that of the control group (P<0.05). These results imply that EsAkt plays an important role in pathogen challenge by regulating the apoptosis related gene expression in E. sinensis, which enriches the basic data of the PI3K-AKT signaling pathway in E. sinensis and lays a foundation for further research on the immune defense mechanism of crustaceans.

Abstract:Pufferfish meat is delicious, nutritious and mostly cooked fresh. In order to prolong the shelf life and enrich pufferfish products, Takifugu obscurus was used as raw material in this study to develop a type of pufferfish sausage. Pediococcus pentosaceus, Lactobacillus plantarum and Staphylococcus xylosus were used as fermenting agents fermentation conditions were optimized by orthogonal experimental design, and the formula was determined by single factor experiment and fuzzy mathematics comprehensive evaluation. The results showed that the optimal supplementary ingredients compoIn conclusion glucose 3%, edible salt 2% and potato starch 10%, and the optimal fermentation conditions were inoculation amount 3.5%, fermentation time 10 h and fermentation temperature 35 ℃. The sensory evaluation score of the optimal fermentation conditions was 79.70. In conclusion, a comprehensive sensory evaluation method based on fuzzy mathematics was established, and the optimal formula and fermentation conditions for fermented pufferfish sausage were determined, which provided a theoretical basis for the development of fermented pufferfish products.