Abstract:Estuaries are the unique ecosystem with variable environment and higher diet foundation, are the spawning grounds, feeding grounds and nursery grounds of many fishery species, which greatly contribute to the fisheries yields. So, estuary ecosystem health is critical to the sustainable recruitment of fishery species. In the present study, based on the projections of fish abundance increment distribution under three climate change scenarios (RCP2.6, RCP6.0 and RCP8.5) in the Yangtze River estuary and Yellow River estuary by the dynamic bioclimate envelope mode, the fishery ecosystem health in Yangtze River estuary and Yellow River estuary was evaluated by hierarchical grey comprehensive evaluation model. The health evaluation system in Yangtze River estuary and Yellow River estuary included environment, fishery community structure and ecosystem function and service. During 2015–2050, the highest health index was found in RCP2.6 scenario both in Yangtze River estuary and Yellow River estuary, and then was RCP6.0 scenario, the lowest health index was found in RCP8.5 scenario. The health level gradually decreased with the increase of greenhouse gas emission levels. The health index showed the “high-low-high” trend with time under RCP2.6 scenario both in Yangtze River estuary and Yellow River estuary; however, it was “low-high-low” trend under RCP6.0 scenario. Under RCP8.5 scenario, the amplitude of health index fluctuation was lower than those under the other scenarios, and there were some differences between two estuaries, the health indices showed a “low-high-low” changing trend in Yangtze River estuary, meanwhile, the health indices firstly increased, then decreased, finally showed a slight increase in Yellow River estuary. If the health index in 2050 was regarded as the final results, the health levels under RCP2.6 scenario were higher than those under the other scenarios in two estuaries. The health index under RCP2.6 scenario was 0.61 in Yangtze River estuary, and it was 1.9 times and 1.8 times for RCP6.0 and RCP8.5 scenarios, respectively. The health index under RCP2.6 scenario was 2.8 times and 2.2 times for RCP6.0 and RCP8.5 scenarios in Yellow River estuary, respectively.

Abstract:Based on the investigation data from 21 sampling stations in the central region of the Bohai Sea in 2012, and by using Nemerow index, eutrophication index (EI), and potential eutrophication assessment model, an assessment was conducted on the environmental quality of water body in the Bohai Sea area. According to the Nemerow index assessment on the 12 indicators including pH, dissolved oxygen (DO), dissolved inorganic nitrogen (DIN), dissolved inorganic phosphorous (DIP), chemical oxygen demand (COD), total petroleum hydrocarbons (TPHs), Cu, Zn, Pb, Cd, Hg, and As, 57.1% of the surface water quality was at slight pollution level or more serious pollution level, 33.3% of the ten meter water quality was at slight and moderate pollution level, 23.8% of the bottom water quality was at slight or more serious pollution level. TPHs was the most important factor in all the contamination factors. Among the 21 surface sea water samples, 12 (corresponding to 57.1%) were above the second marine water quality standards when evaluated with single factor index method. The standard-exceeding rates of ten-meter water and bottom water were 42.9% and 14.3% respectively. The polluted area was located in the southeast of survey area, next to the Bohai Bay and the Laizhou Bay. The horizontal distribution of sea water quality showed an increasing trend from the coastal area to the middle area. The sea water EI value of the central region of the Bohai Sea except 13 stations in the surface water was less than 1, the results showed that most of the seawater quality in the investigated area was at the poorly eutrophication level. The ratios of N/P in most of the sea water were much higher than Redfield ratios, and the situation of nitrogen limiting in the central Bohai Sea was gradually changing to that of relative lack of phosphate. The nutrients structure has changed into potential eutrophication of phosphorus limiting.

Abstract:According to the monitoring results of the surface sediment samples from five cruises of 150 survey stations from 2006 to 2014 collected in the adjacent sea water of the Yellow River estuary, the pollution status and the annual variations of the seven kinds of heavy metals Hg, Cu, Pb, Cd, Cr, Zn and As in the sediments were analyzed. The methods of single factor standard index and potential ecological risk index were used to evaluate the status of heavy metal pollution and potential ecological risk degree. The results showed that the contents of heavy metals met the first class standard requiement of the Marine Sediment Quality except a few stations investigated in 2011 exceeded the first class standard. The contents of Hg and Cd were equal, which were obviously lower than that of the other five heavy metal elements. And the annual variations of the seven heavy metals were different from each other. The average single factor standard indices of the seven heavy metals were all less than 1, which indicated that the quality status of the surface sediment in the survey area was good. The order of the annual average single factor standard indices of the seven heavy metals was Cu>Cr>As>Pb> Cd>Hg>Zn. And the standard index value of Cu was higher than the others, even the average standard index of Cu reached 0.61 in recent ten years, which should be paid attention to and strengthened routine monitoring. And the single factor standard indices for the other six heavy metal elements were all less than 0.4. Further potential ecological risk assessment results showed that the average potential ecological risk factors of seven heavy metals of all cruises were less than 40, which presented a low level ecological risk. And the order of for seven heavy metals in recent ten years was Hg>Cd>As>Pb>Cu>Cr>Zn, where Hg was the main potential ecological risk factor in the survey area. The annual variations of the comprehensive potential ecological risk index RI ranged from 32.18 to 53.12, which indicated a low level of ecological risk. Basically, the trend of the annual variations of RI was increasing first, then decreasing, and then a slight increasing.

Abstract:In order to improve the fishing techniques and management level of offshore fishery resources, promote the implementation of National Marine Fishing Gear Catalog, selectivity among single gillnet (with the mesh sizes 40 mm, 50 mm, 60 mm), double gillnet, trammel gillnet and productive fishing gear (control group) was compared in the Liaodong Bay during October to November 2014. The results showed that the catch species of all experimental and productive fishing gear was between 6 and 9; the dominant species of experimental and productive fishing gear were Oratosquilla oratoria (dominance decreased with the increased mesh size) and Lateolabrax japonicas (dominance increased with the increased mesh size). There were significant differences on yields of the same type nets with different mesh size (P=0.0003, P=0.001, P=0.002), compared the CPUE of 60 mm with 40 mm, the single gillnet is 800%, the double gillnet is 650% and the trammel gillnet is 500%; but the influence on catch yields of the different type nets with same mesh size was relatively small. The average similarity of intra-group catch composition of the network was: single gillnet 59.24%, double gillnet 62.63%, and trammel gillnet 66.51%; Meanwhile, the average similarity of the inter-group was 71.44%, 67.50% and 70.58% respectively; The species which had the highest contribution to intra-group and inter-group average similarity were O. oratoria and L. japonicas. With the increasing of mesh size, the proportion of the catch the proportion of juvenile in the catch of all types of gillnets showed a decreasing trend.

Abstract:The transcription factor c-Jun (jun proto-oncogene) of AP-1 family and its role in immune response is unknown in half-smooth tongue sole (Cynoglossus semilaevis) so far. Based on c-Jun genetic partial cDNA sequence of half-smooth tongue sole, the complete cDNA sequence is obtained by RACE and PCR application. The cDNA of c-Jun is 2093 bp in length, including a 377 bp 5¢-terminal untranslated region (UTR), a 735 bp 3¢-terminal UTR and a 981 bp open reading frame (ORF), encoding a polypeptide of 326 amino acids. The deduced amino acid sequence of c-Jun possesses a Jun domain which is a typical structure domain in AP-1 family and a high conservative leucine zipper structure domain (BRLZ). The protein sequence alignment and phylogenetic tree analysis suggested that the half-smooth tongue sole c-Jun gene is closely related to that of the rainbow trout’s (Oncorhynchus mykiss). Tissue expression analysis by quantitative real-time reverse transcription PCR showed that the c-Jun mRNA was constitutively expressed in all examined tissues with the predominant expression in the ovay. After infection with Vibrio anguillarum, the half-smooth tongue sole c-Jun transcript levels exhibited distinct time-dependent response patterns in the liver, spleen, head kidney, intestine, gill and blood. Among the tested tissues, changes in gill is the most obvious, and it reached the peak level 13.20-fold at 12 h post-infection. After challenge with different pathogenic (LPS, PGN, PolyI:C, WGP) in lymphocyte, the c-Jun gene in the WGP group showed up-regulation, while in the LPS group and PolyI:C group it showed down-regulation, while, there was no significant change in the PGN group. The results suggested that the c-Jun gene plays a vital role in the immune response in half-smooth tongue sole and might play an important role in half-smooth tongue sole host defense against the bacteria infection.

Abstract:Platichthys stellatus and Kareius bicoloratus are two common Pleuronectidae species that are highly valuable in the aquaculture industry. P. stellatus is endowed with high meat content and delicious taste while K. bicoloratus is highly resistant to diseases. Characteristics of their hybrids, however, were rarely reported. In this study, we explored the genetic variability of mtDNA sequences of P.stellatus, K.bicoloratus and their hybrid (P.stellatus♀×K.bicoloratus♂), using 24 P. stellatus, 24 K. bicoloratus and 40 hybrids as samples. We found that that the content of A+T base pairs was higher than that of the C+G base pairs in all 3 groups, and that the content of A+T base pairs in the hybrids was the same as in P. stellatus. The analysis of mtDNA Cytb gene and COI fragments suggested that the genetic distances between K. bicoloratus and P. stellatus were 0.085 and 0.045 respectively, and that the genetic distances between the hybrids and K.bicoloratus were 0.076 and 0.045 separately. Moreover, there was a very small differentiation (0.009) between the hybrids and P. stellatus based on the mtDNA Cytb sequence analysis, and there was no genetic differentiation between the 2 groups based on the mtDNA COI gene sequence analysis. The neighbor-joining (NJ) phylogeny trees constructed with the Kimura 2-parameter model showed that P. stellatus and the hybrid could be clustered into one branch, while K.bicoloratus belonged to another branch. In the NJ phylogenetic tree based on gene COI, the big branch including F1 hybrid and P. stellatus could be further divided into two small branches. One was P. stellatus and F1 hybrid with the left eyes, and the other was the F1 hybrid with the right eyes. These data strongly suggested that characteristics of the hybrid of P.stellatus and K.bicoloratus (P.stellatus♀ × K.bicoloratus♂) were maternally inherited. This study provided helpful information for the future research on the hybridization of the two species.

Abstract:Eleutheronema tetradacty is a tropical inshore marine fish species that belongs to the Polynemidae family. It has very high commercial values. Previous studies mainly focused on the stock structure and taxonomy of this species. However, the information about morphology and structural and functional biology has been limited, especially little is known about the development of the digestive system. In this study we applied the histological method to investigate this issue of E. tetradactylum aged Day 1 to Day 30 after hatching (DAH 1 to 30). Thus we may develop a better feeding strategy during artificial seedling rearing and improve the survival rate of the larvae. The experimental conditions were temperature 29–32℃，salinity 25–28, dissolved oxygen 4.80–5.5 mg/L，and pH 8.0–8.3. We found that on DAH 1, the digestive tube was not differentiated, and was composed of intestinal tubes that were attached to the abdominal wall and the yolk sac. On DAH 3, the yolk sac was fully absorbed, the larvae began feeding, and the digestive tube started to communicate with the outside. This indicated a switch from the endogenous nutrition to the exogenous feeding, and from this point, the larvae could maintain the growth by relative independent digestion and absorption through the gradually differentiated esophagus, stomach and intestine. The gastric gland and pyloric caeca appeared on DAH 15, which greatly improved the digestion and were the signs of the juvenile stage. On DAH 30, the structure and function of the digestive tract were well developed, which resembled that of the adults. The liver and the pancreas started to differentiate on DAH 2 and DAH 5 respectively, and they developed fast as the larvae grew. These observations suggested that the development of the digestive system was highly associated with the functions. Based on the characteristics of the digestive system, we further discussed issues in three major critical stages in the process of seedling rearing and proposed solutions for each stage.

Abstract:Ammonia is a major source of water pollution and a cause of fish diseases. The object of this study was to evaluate the effects of ammonia-N stress on the gill Na+/K+-ATPase, the liver microstructure, and serum physiological-biochemical indices of juvenile Verasper variegatus. Experimental fish had an initial average weight of (70.0±5.5) g. Subjects were first exposed to ammonia-N for 96 hours to obtain the median lethal concentration at 96 h. According to the data, we set up a control group and three ammonia-N treatments of which the concentrations were 35 mg/L (low group), 50 mg/L (middle group), and 65 mg/L (high group). In each group the subjects were treated for a series of periods (0, 6, 12, 24, 48 and 96 h). In the gill microstructure, the ammonia-N stress caused changes such as the increase in chloride cells, the shedding of pavement cells, epithelial tissue hyperplasia, the fracture in blood capillaries, the overflow of red blood cells, shortening and curling of thickened gill dice, and the breakage and congestion of the gill cavity. In liver tissue, we observed dissolution of liver nuclei, vacuoles in cells, liver cell degeneration, liquidized cells, blood sinus expansion, hyperemia, and severe congestion. Under the ammonia-N stress the activities of Na+/K+-ATPase and glutathione peroxidase (GSH-PX) were first decreased and then increased, which was significantly different from the control group (P<0.05). The superoxide dismutase (SOD) activity was raised followed by a decrease, and was higher than the control group (P<0.05). After 96-hour treatment with ammonia nitrogen, the malondialdehyde (MDA) concentration became higher than the control group, and there was a positive correlation between the MDA concentration and ammonia nitrogen concentration (P<0.05). Ammonia nitrogen stress also resulted in an increase-decrease change in activities of serum alanine amino transferase (ALP), aspartate amino transferase (AST) and lysozyme (LSZ) (P<0.05). After 96-hour treatment, activities of liver ALT and AST first increased and then decreased, and was significantly lower than the control group (P<0.05). The liver LSZ activity was significantly lower than the control group after 96-hour treatment (P<0.05). These results suggested that ammonia nitrogen stress could cause a variety of impairments in the fish body, including deterioration of the antioxidant system, nonspecific immune system, physiological metabolism, the gill tissue, the respiratory function and the detoxification function. Also it led to the liver tissue hyperemia and the formation of dot lesions.

Abstract:Low temperature tolerance of Epinephelus moara♀× E. lanceolatus♂(hereinafter Yunlong) was investigated in this study. The initial water temperature (20℃) was served as control, and then was dropped at a rate of 1℃/d, the changes in serum biochemical indices and metabolic enzyme activities of Yunlong were determined when the water temperatures reached 16℃, 15℃, 13℃ and 10℃. During the cold stress period, survival rates and the semi-lethal temperatures of Yunlong, E. fuscoguttatus♀× E.lanceolatus♂ (Pearl Gentian), and E. coioides♀ × E. lanceolatus♂(Qinglong) were observed and recorded. The semi-lethal temperature was recorded when the survival rate reached 50%. We found that the semi-lethal temperatures for Pearl Gentian, Qinglong and Yunlong were 11℃, 9.5℃, 9℃, and 8℃ respectively. The levels of serum creatinine (CREA) and total cholesterol (T-CHO) were first increased and then decreased along with increase of the intensity and duration of the low temperature stress. The level of serum triglycerides (TG) fluctuated at different temperatures, and the values at 15℃ and 10℃ were significantly different from that of the control group (P<0.05). The content of serum glucose (GLU) also varied with the temperatures, the levels at 16℃ and 13℃ were different from that of the control group (P<0.05). The activity of serum alkaline phosphatase (AKP) at 16℃ was slightly and insignificantly increased (P>0.05). The activity of serum catalase (CAT) fluctuated and showed significant differences between the control group and low temperatures at 16℃, 15℃ and 10℃ (P<0.05). The activity of serum glutamic-oxaloacetic transaminase (GOT) was slightly higher than that of the control group (P>0.05). The activity of serum glutamate pyruvate transaminase (GPT) was elevated followed by a decline along with the increase in the intensity and duration of low temperature stress. At 16℃ and 15℃, the activity of GPT was different from that of the control group (P<0.05). In conclusion, Yunlong was the most tolerant to low temperature stress among the three hybrids. Our data also suggested that low temperature could impair the immunity and the antioxidant capacity of juvenile fish, therefore the intensity and duration of the low temperature condition should be limited during practice.

Abstract:Here we investigated the effects of salinity gradients (7, 12, 17, 22, 27, 32, 37, 42, 47, and 53) on the survival, the first feeding and feeding of larval Centropristis striata. The first feeding rate (FFR%), the feeding rate and the feeding intensity of early larvae were also recorded and analyzed. Newly-hatched larvae were used in the starvation test to measure the survival activity indices (SAIs) at different salinities. The results suggested that salinity had strong effects on the survival and feeding of larvae. The suitable salinity for newly- hatched larvae was 12–37, and the optimum range was 27 to 32. The survival rate, the first feeding rate and the feeding intensity were reduced when salinity was below 12 or above 37. The SAI of newly-hatched larvae varied between 0.034 and 6.401 according to the salinity. When the salinity was between 12 and 37, the corresponding SAIs were significantly different from those at salinities 7, 42, 47 and 52 (P﹤0.05). The SAI was the highest (6.041) at salinity 32. The relationship between SAI (y) and salinity (x) could be described as y = –5.894+1.155x–0.034x2+0.000x3, R2 =0.895. When the salinities were 7, 42, 47 and 52, the survival rate of larvae was below 50%. There was no survival after a 3-day treatment at salinity 52, or after 4 days at salinity 47. After a 5-day treatment at salinities 22, 27 and 32, the survival rate was above 80%. At salinity 17 to 37, the first feeding rate varied between 60% and 85%, and the maximum (85%) appeared at salinity 32. The highest feeding rate (85%) was also observed at this salinity, and the feeding intensity was 5.45 rotifers per larvae on Day 8.

Abstract:This experiment used laboratory-made fish protein hydrolysates (PHf) and the commercial chicken protein hydrolysate (PHc) to replace 10% total fish meal protein and designed three isonitrogenous and isoenergetic diets: the control group (FM), PHf group (fish protein hydrolysate) and PHc group (chicken protein hydrolysate). Juvenile turbot (Scophthalmus maximus L.) (4.16±0.01 g) were fed with these three diets for 12 weeks to investigate the effects of different protein hydrolysates on the growth performance and intestinal structure of juvenile turbot. Results of this study showed that the specific growth rate, feed efficiency, protein efficiency ratio and protein retention in FM group and PHf group had no significant difference (P>0.05), but both the two groups were significantly higher than the PHc group (P<0.05); there was no significant difference in feeding rate in the three groups (P>0.05). PHf and PHc had higher crude protein than FM (P<0.05), while had no significant difference in crude lipid (P>0.05). PHf and PHc had higher essential amino acid content than FM (P<0.05). The plica height in foregut and midgut of PHf and PHc were higher than FM, and PHf had significant difference with FM (P<0.05), while the intestinal wall thickness of FM was higher than the other two groups, and significantly higher than PHf (P<0.05); there was no significant difference in foregut and midgut enterocytes height of the experimental groups (P>0.05). These results indicated that the growth response and feed utilization of PHf was superior to PHc, which suggesting that replaced fish meal by fish protein hydrolysates in high plant protein diets had some advantage over chicken protein hydrolysates.

Abstract:Large yellow croaker Larimichthys crocea is one of the most successful marine fish culture operations in terms of the number of juveniles produced and commercial size fish production annually in China. The production of this farmed species was more than 120000 metric tons in 2014 in China. However, large yellow croaker was often encountered with many stress situations, such as net cage changing, typhoon and water temperature oscillations during the grown-up farming. Generally, these could result in the decreases of feed utilization, anti-diseases ability and growth, even causing great economy losses. So, it is important to improve the anti-stress ability of large yellow croaker. The purpose of this study was to investigate the effects of dietary glycine on anti-oxidative and anti-stress responses of large yellow croaker (initial body weight 130.35±8.37 g). There were 6 experimental diets containing graded levels of glycine (1.58%, 2.15%, 2.75%, 3.96%, 6.33% and 7.51%) with different levels of glycine supplementation (0, 0.6%, 1.2%, 2.4%, 4.8% and 6.0%). After a 30 d feeding trial, the experimental animals were challenged with a 30 s trawl stress for three times. The results showed that the survival and body compositions of large yellow croaker had no significant differences among all the treatments (P>0.05). The significant highest value of the total anti-oxidative capacity in liver was found in the treatment with 2.75% dietary glycine. The lowest value of the malondialdehyde in liver was found in the treatment with 3.96% dietary glycine. However, this value of malondialdehyde did not significantly differ from that in the treatment with 2.75% dietary glycine (P>0.05). On the basis of hepatic total anti-oxidative capacity, using the second-order regression analysis, the most reasonable dietary glycine content for large yellow croaker was estimated to be 3.57%. Both the glutamic-oxaloacetic transaminase and the glutamic -pyruvic transaminase activities in serum had the lowest value in the treatment with 2.75% dietary glycine (P<0.05). Before the trawl stress, treatment with 2.75% dietary glycine resulted in the highest concentration of cortisol in serum (P<0.05). After the trawl stress, the serum cortisol concentration in this group kept little change. Meanwhile, the glucose concentration in serum kept at a relative higher level. In conclusion, for the purpose of improving the capacities of anti-stress in large yellow croaker, while had no significant negative effects on the survival and liver health, 2.75%–3.57% of dietary glycine were recommended for large yellow croaker.

Abstract:This experiment was conducted to study the effects of dietary bile acids levels on growth, muscle composition and digestive enzyme activities of juvenile Ctenopharyngodon idellus. Five hundred and forty healthy fish, mean body weight of (13.11±0.58) g, were randomly selected and divided into six groups, with three replicates in each group (30 fish/replicate). Each group was fed a diet containing bile acids level of either 0 (control group), 75 mg/kg, 150 mg/kg, 225 mg/kg, 300 mg/kg and 375 mg/kg for a period of 60 days. The results showed that the increase of dietary bile acids levels resulted in an initial elevation of weight gain rate (WGR), specific growth rate (SGR) and followed by a decrease in WGR and SGR when bile acids was 375 mg/kg in the diet. Furthermore, inclusion of 300 mg/kg dietary bile acids produced the highest WGR and SGR (281.18% and 2.23%/d respectively). By contrast, coupled with the increase of dietary bile acids, the feed conversion ratio (FCR) decreased initially and then increased. The FCR reached the lowest (1.26) level when the dietary bile acids level was 225 mg/kg. Based on square regression analysis, to optimize each growth parameter: WGR, SGR and FCR during C. idellus juvenile growth, the most appropriate inclusion levels of dietary bile acids were 222.15 mg/kg, 265.00 mg/kg and 242.86 mg/kg respectively. The crude lipid of muscle during the experiment decreased initially and then leveled out, while the crude protein of muscle increased with the increase of dietary bile acids levels (0–300 mg/kg) and then decreased. Similarly, digestive enzyme activities of segments intestinal and hepatopancreas had a tendency of increasing with the elevated bile acids inclusion and then leveled out at the end. Results indicated that certain range of dietary bile acids levels could promote growth, fat metabolism, reduce fat deposition in muscle and improve meat quality. In conclusion, the bile acids supplementary level of 222.15–265.00 mg/kg is recommended for growing juvenile C. idellus based on the consideration of digestive enzyme activities, meat quality and growth performance.

Abstract:Fatty liver was a common disease in aquaculture. In recent years, the occurrence of fatty liver in fish has significantly increased, but the mechanism of this disease remains unclear. Most researches have focused on the pathogenesis of the disease, while little is known at the cellular level. In this study, we used primary hepatocyte culture of Jian Carp (Cyprinus carpio var. Jian) as the experimental material, and induced the hepatocyte injury by optimal exposure (dose and time) to oleic acid. The hepatocytes of Jian Carp were isolated and purified with trypsin digestion and then were cultured in a medium. To identify the proper concentration of oleic acid, the hepatocytes were treated with a gradient of concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8 mmol/L) for 24–48 h, and the supernatants and hepatocytes were then collected at different time points. We measured the contents of multiple biochemical parameters in the collected samples, including triglyceride (TG) and the total cholesterol (TC), as well as the activities of enzymes such as alanine aminotransferase (GPT), aspartate aminotransferase (GOT), γ-glutamyltranspeptidase (γ-GT), lactate dehydrogenase (LDH), and superoxide dismutase (SOD). We also determined the survival rate of hepatocytes using the MTT method. Lipid droplets in the cells were observed using the oil red staining method. Our results suggested that the cultured hepatocytes grew properly with a normal morphology, and that the hepatocytes could be used for the study of lipid metabolism. After the oleic acid treatment at different concentrations of oleic acid for 24–48 h, we found that the exposure to 0.4 mmol/L oleic acid for 48 h significantly elevated the levels of GOT, GPT, LDH, TG,TC, and γ-GT, but reduced the activity of SOD (P﹤0.05 or P﹤0.01). Lipid droplets were detected in the hepatocytes under light microscope after the treating with 0.4 mmol/L oleic acid for 24–48 h. The biochemical indices described above indicated that 0.4 mmol/L oleic acid and 48 hours could be the optimal conditions to induce hepatocyte steatosis in cultured hepatocytes. Therefore, in this study we successfully established a hepatocyte steatosis model that will be a useful tool in the future study of fatty liver disease in fish.

Abstract:An 8-week feeding experiment was conducted to study the effects of different feeding levels on growth performance, intestinal digestive enzyme activities and serum physiological and biochemical indices of Asian swamp eel (Monopterus albus) at two different sizes [mean initial body weight: big-size (68.85±0.44 g) and small-size (26.67±0.17 g)]. There were four big-size groups which had 4 feeding rates (2.8%, 3.6%, 4.4% and 3.6% body weight/day); And five small-size groups which had 5 feeding rates (3.0%, 4.0%, 5.0%, 6.0% and 7.0% body weight/day). The stocking densities were 50 fry/4500-litre tank of big-size groups and 100 fry/4500-litre tank of small-size groups. Each group had three biological repeats. The results showed that with the increase of feeding levels for the two sizes of swamp eel, the weight gain rate, feed coefficient, liver body rate, and content of fat in the whole fish were significantly higher (P<0.05), but the content of protein in the whole fish had a decreasing tendency. When the feeding level was 4.4% and 5.2% for big-size swamp eel, the weight gain rate reached a peak, and there were no significant differences between the two groups. The activities of intestinal lipase and trypsin were significantly increased (P<0.05) with the increase of feeding level; serum superoxide dismutase (SOD) activity and total cholesterol (TC) content showed a trend from rise to decline; serum triglycerides (TG) content was rising. When the feeding level was 4.4%, the activities of SOD and lysozyme (LZM) reached the highest level among all the groups of big-size swamp eel. In pace with the rising of feeding level for the small-size groups, intestinal trypsin activity was increased significantly (P<0.05); the content of serum TG and TC rose gradually; the SOD activity was decreased; the content of blood sugar had a fluctuated trend. Compared with other groups of small-size swamp eel, the activities of SOD and LZM had higher standards when the feeding level was 6%. In conclusion, when the feeding level were 4.4% for the big-size and 5%–6% for the small-size respectively, the growth performance of swamp eel reached a better status.

Abstract:Misgurnus anguillicaudatus is a fish species with high economical values, and is widely distributed in China except for the Qinghai-Tibet Plateau. The popularity of this fish has been growing due to its high contents of minerals, amino acids and vitamins. Environment is one of the main factors that affect the body color, the texture and the visceral enzyme activities of the fish; hence it has been a hotspot in the research of M. anguillicaudatus. In this study we explored the living strategies of M. anguillicaudatus raised in two culture models, the pond monoculture (M1) and the polyculture of turtles, fish and shrimp in the rice field (M2). The wet earth from the paddy field served as the control (M0). We analyzed the differences in the body color, the texture, digestive enzymes, antioxidant enzymes and ATP enzymes between the two populations. The average body length and weight of the samples were (113.37±4.55) mm and (13.35±1.23) g for M1, and (122.69±5.22) mm and (16.43±1.36) g for M2. SPSS 19.0 and Excel 2003 were employed for data analysis. The results were as follows: (1) The measures of the dorsal chromatism of adult M. anguillicaudatus were M0>M1 (P<0.05). (2) There were significant differences in the measured indicators of the textures of dorsal muscles between the two populations, and all the values followed the order M2>M1 (P<0.05) except for that of adhesiveness. (3) There was no difference in the activities of the visceral digestive enzymes except for protease (M2>M1, P<0.05) and lipase (M1>M2, P<0.05). (4) Activities of antioxidant enzymes (CAT, SOD and POD) of M2 were higher than those of M1 (P<0.05). There were significant differences in activities of hepatopancreas enzymes between the two populations (P<0.05). The activities of Ca2+-Mg2+ATPase and Na+-K+ ATPase of M2 were significantly higher than those of M1 (P<0.05).

Abstract:This study was designed to explore whether the heat-inactivated WSSV could induce immune priming response on Fenneropenaeus chinensis. During the initial 6 days, four experiment groups (E15℃, E23℃, E28℃, and E32℃) were fed with typical symptoms of WSSV prawn muscle, which was dealt with at 60℃ for 1 h using the equivalent and quantitative test method. At the same time, we set a positive control group C23℃ (fed with the unheat-inactivated WSSV muscle) and a control group CF23℃ (fed with commercial compound feed only). At the 13th day of the experiment, all shrimps were infected with WSSV again. The result showed that 60℃ could decrease the activity of WSSV absolutely, there were no dead shrimp in the initial 12 d, but the mortality of positive control group C23℃ was 100%. The survival rates of different experiment (E15℃, E23℃, E28℃, and E32℃) and control groups were 80.41%, 33.29%, 8.47% , 16.43% and 8.89%, respectively, after 19 days. The survival rate of E15℃ showed highly significant difference from other experiments (P<0.01). In addition, there was a significant difference between E23℃ experiment group and other experiment groups (P<0.05). Nevertheless, there was no significant difference between E28℃ and E32℃ groups (P>0.05). Compared to the results of absolute quantitation, it was found that the multiplication rate of WSSV was the highest under 28℃, high (32℃) and low (15℃) temperature would inhibit the multiplication of WSSV in the shrimps which accepted the subsequent rechallenge of WSSV after the heat-inactivated WSSV induced. Experimental results showed that heat-inactivated WSSV could induce immune priming response and provide some protection to the infected shrimp by WSSV. The multiplication rate of WSSV is closely related to temperature.

Abstract:Atrina pectinata is a large deep-water mollusk species that has high economic values. It is distributed in the coastal areas of China, from the Liaodong Peninsula in the north to the Qiongzhou Strait in the south. Its habitat is adjacent to China’s provinces such as Fujian, Guangdong, Liaoning and Shandong. In recent decades, the natural resource of A. pectinata has declined due to the environment destruction and overfishing. To better protect the resource of A. pectinata, we need to understand its population genetic structure. Microsatellites is a widely used method to assess the genetic diversity in farmed aquatic species and construct QTL due to its characteristics such as the abundant polymorphism, the rich information, the co-dominance and conservation. The EST-SSR marker is inexpensive and is probably associated with functional regions of the genome. Therefore, in this research, we developed a series of EST-SSR markers using a transcriptome-based platform to study the genetic diversity of A. pectinata. We identified 10550 EST-SSR (8.2%) using MISA software, and the corresponding frequency was 1 EST-SSR per 9.01 kb of the sequence. Dinucleotide repeats were dominant among all EST-SSRs, counting for 77.08%. We designed 120 primers for PCR, and 36 out of 120 resulted in successful amplification. Fragments amplified with 12 primers were polymorphic. Next we used these SSR primers to explore the genetic variation of 30 A. pectinata samples collected from the Qingdao Bay. The number of alleles for the 12 SSR makers varied from 2 to 5 in these samples with an average of 3.5 alleles per locus. The observed heterozygosity ranged from 0.1667 to 0.6667, and the expected heterozygosity varied from 0.4316 to 0.7938. Polymorphic information content ranged from 0.3679 to 0.7459. These indicated high genetic diversity of the A. pectinata population in the Qingdao Bay. Moreover, we verified that the polymorphic EST-SSR markers could be a useful tool in comparative mapping, gene tagging and QTL mapping.

Abstract:To investigate the effects of acute hypersaline stress on the physiological metabolism and immunity of clam Trachycardium flavum. The oxygen consumption rate, ammonia excretion rate and activities of Na+/K+-ATPase, superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (ALP) in different tissues were measured at 2, 12, 24, 48 and 72 h as water salinity increased from 31 to 37. Results showed that the oxygen consumption rate of clams in treatment group decreased at 2 h and then gradually increased, and was significantly higher than the control group at 24 and 72 h (P<0.05). The ammonia excretion rate significantly decreased (P<0.01) at 2 h and was lower than the control group at all time points. At 12 h, the clams showed significant lower Na+/K+-ATPase activity (P<0.05), but no significant difference of Na+/K+-ATPase activity in muscle was found between the control and treatment groups. SOD activities in mantle, hepatopancreas and gill reached maximum values at 24 h, which was significantly higher than the control group(P<0.05), and then decreased; SOD activity in muscle reached a peak at 48 h and then declined. ACP activity in mantle and gill showed similar variation as SOD. ACP activity was significantly reduced (P<0.05) and then gradually rose. No significant difference of enzymatic activity in muscle was found between the control and treatment groups. ALP activity in hepatopancreas and gill decreased followed by significant increase (P<0.05). Compared with the control group, no significant difference of enzymatic activity in mantle and muscle was found. The results suggested that acute hypersalinet stress significantly affected the metabolism and immunity of T. flavum, and such effects varied with time. The impact of hypersaline on the immune enzymatic activities is tissue-specific.

Abstract:Rock bream (Oplegnathus fasciatus) is an important economic fish cultured in China. In recent years, rock bream aquaculture has developed rapidly along the coast of China. In August 2013, an epizootic occurred among cultured rock bream in a cage culture farm in Zhoushan of Zhejiang Province, China, which caused a cumulative mortality up to 20%. The diseased fish had no obvious symptoms on body surface; the main symptom was characterized by many white nodules (1–2 mm) in the spleen and kidney. The aim of this research was to isolate and identify the bacterial isolate, then to determine the pathogenicity and test drug sensitivity of bacteria pathogen found in diseased rock bream. A dominant bacterium strain OF-1 was isolated from the liver, spleen and kidney. Artificial infection test with intraperitoneal injection method proved that the LD50 value of bacterium strain OF-1 was 5.93×104 CFU/g. It was identified based on the morphology, the biochemical characteristics, and the 16S rRNA sequencing. The results of morphological tests indicated that strain OF-1 was gram-negative, rod bacteria and did not grow in the TCBS plate. OF-1 exhibited an API 20E index profile of 2005004, which was identical with the profile exhibited by Photobacterium damselae subsp. piscicida. The sequence analysis of 16S rRNA gene, the morphological, physiological and biochemical characteristics indicated that the bacterium strain OF-1 could be identified as P. damselae subsp. piscicida. The strain was highly sensitive to gentamicinum, penicillin G, norfloxacin, ofloxacin and ampicillin, but was resistant to erythromycinum, vancomycin, rocephin, streptomycin, kanamycinum and proctaphlin. This paper reported for the first time that P. damselae subsp. Piscicida caused the disease of rock bream in China, which will provide reference in fish health management and disease control.

Abstract:Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office internaptional despizooties) Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 (100%) L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 (49.1%) L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 103–107 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 103–105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×104 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×104 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.

Abstract:Portunus trituberculatus, aka the swimming crab, is an important fishery species with high commercial value in China. They mainly inhabit the sandy and muddy bottom underneath the seawater in the coast of countries such as China, Japan and Korea. The growth and development of P. trituberculatus was characterized by periodic molting activities in which the new ectoskeleton grows while the old one molting. Previous studies reported that chitinase was an essential enzyme involved in the molting of crustaceans, and that it was required in chitin digestion and the immunity to the pathogen infection. However, little has been known about the effect of chitinase on molting activities, and the expression of the enzyme during rapid salinity stress is also obscure. To understand the biological function of PtCht3 gene in P. trituberculatus, we cloned and analyzed the full-length cDNA sequence of PtCht3 gene using SMARTTM RACE amplification kit. The cDNA sequence had 1409 bp encoding 394 amino acid residues. The isoelectric point (pI) of the putative peptide was 4.80, and the predicted molecular mass was 43.67 kDa. PtCht3 could be a stable protein with the hydrophilic coefficient total average of –0.097. The homology and systematic evolution analysis revealed that PtCht3 of P. trituberculatus was highly homologous with the protein in other species; for example, it shared 54% similarity with Pandalopsis japonica and 53% similarity with Eriocheir sinensis. The phylogenetic analysis showed that P. trituberculatus PtCht3 was in the same class as other arthropods¢ PtCht3. The expression of PtCht3 in different tissues was analyzed with quantitative real-time PCR. We found that the highest expression level appeared in hepatopancreas, and that the expression was significantly up-regulated in the prophase of the molting cycle. In response to the low salinity stress the expression level fluctuated in gills and hepatopancreas, showing a general up-and-down pattern. Our results suggested that PtCht3 might play an important role in digestion and molting activities, and it could be involved in the regulation of osmotic pressure in P. trituberculatus.

Abstract:Epitheliocystis is a kind of fish disease which is caused by bacteria in the order of Chlamydiales. Its typical characterization is epithelial hypertrophy in gills and skin of diseased fish. Fish epitheliocystis has not been reported in China. As a newly cultured marine fish species, spotted knifejaw (Oplegnathus puncatus) has high economic value. O. puncatus juveniles of 15 cm in total length died continuously in a hatchery located in Shandong Province in 2015. And the cumulative mortality was more than 40% in 15 d. The field investigation recorded that the water temperature was 21℃ and the water salinity was 30 during epidemics. Diseased O. puncatus usually scattered in the tank. They stagnated in the water with little vitality and/or lost balance even drifted with the water flow. Clinical signs of diseased fish contained debility, anorexia and dyspnea. Diseased fish kept mouth open with operculum opening and closing at a high rate. They did not response to feeding. Clinical examination and necropsy revealed that gills of diseased fish were covered by a lot of mucus. The gill filaments were damaged and the intestinal tract was empty. Many cysts with 30–70 μm in diameter, round or ovoid in shape and light yellow-brown in color were observed on wet mounts of gill filaments from diseased fish under the light microscope. The histopathology by hematoxylin and eosin staining indicated that the ends of secondary lamellae adhered each other and many epithelial cells enlarged and formed cysts. These cysts were basophilia and homogeneous. The examination of scanning electron microscope showed that the gill filaments of diseased fish were clavate. The secondary lamellae were covered by a lot of mucus and embedded by many cysts with smooth surface. Based on the field investigation, clinical examination and necropsy of diseased fish and microscopic observation of gills, this disease was considered as epitheliocystis of O. puncatus. This is the first report of epitheliocystis in cultured O. puncatus in China.