Abstract:In this study, based on the water nutrients data of Sanggou Bay surveyed in the autumn of 2014, and the spring, summer and winter of 2015, the seasonal variation and distribution of nutrients were analyzed and the nutrient levels were evaluated. The results showed that dissolved inorganic nitrogen (DIN) levels in the autumn of 2014 was the highest among four surveyed seasons. The average content of NO2-N and NO3-N was the highest during the summer and autumn, respectively. By comparison, the average contents of NH4-N and PO4-P were the highest during the spring and winter, respectively. Compared with the seawater quality standard, DIN contents in four seasons were lower than the levels in the first level of seawater standard. Active phosphate contents were lower than the first level of seawater standard in spring and summer, but were higher than first level of seawater standard in autumn and winter. From the perspective of horizontal distribution, the content of DIN of the inner bay was lower than that of the outer bay in spring, but was higher than those of the outer bay in summer and autumn. The content of DIN exhibited an increasing trend from the north to the south area during the winter. The content of PO4-P showed an increasing trend from the outer bay to the central area in spring and winter. Furthermore, the content of PO4-P showed a decreasing trend from the outer bay to the central area in summer, and the content of PO4-P of the inner bay was higher than that of the outer bay in autumn. The N/P ratio was the same as the Redfield ratio in autumn of 2014, and the N/P ratio was higher than the Redfield ratio in spring, summer and winter of 2015, with relatively low levels of inorganic phosphorus. According to the potential eutrophication assessment model, nutrition levels were relatively poor in the autumn of 2014, and the spring, summer and winter of 2015. Compared with the 1980s and 1990s data of Sanggou Bay, the main limiting factor for phytoplankton growth has changed from nitrogen to phosphorus limitation in spring and summer.

Abstract:The big-eye tuna (Thunnus obesus) is the most commercially important tuna species in eastern Pacific Ocean. The fishery of this species has been managed by Inter-American Tropical Tuna Commission (IATTC). IATTC evaluates the stock of big-eye tuna annually using Stock Synthesis 3 (SS3). SS3 is one of the most commonly used models that assess age-structured fishery stock based on biological parameters and fishery data such as the annual catch, the age-composition, the length composition of catch and abundance indices. A recent assessment of the big-eye tuna stock in eastern Pacific Ocean indicated a small recovery of the spawning stock biomass (SSB) and the fishing mortality (F) with respect to biological reference points (BRPs). The integrated model effectively combines multiple data with various sources into single analysis, and propagates the uncertainties associated with model parameters and structures into model outputs such as the biomass time series and BRP. However, it is time-consuming to use full SS3 model to analyze the big-eye tuna stock as it has as many as 23 fisheries. Therefore, model simplification has become necessary to improve the analysis efficiency. In this study, we tried to simplify the SS3 modeling process by using 2 fisheries (purse seine and longline fisheries) instead of 23. Representative abundance index and size composition from the purse seine fisheries and longline fisheries of the full model were used in the simplified one. The outputs of the simplified model were compared to that of the full model to evaluate the performance of the former. The results showed that the simplified model effectively captured the historical trends of stock time series (e.g., recruitments, SSB and F) and estimated FMSY with little interference from the steepness and natural mortality parameters. The reduced interference from steepness allowed the simplified model to correctly analyze the stock status based on the Kobe plot. However, the simplified model increased errors in the estimation of other BRPs. This study implied that selecting BRPs and defining stock status of big-eye tuna could affect the evaluation of the performance of the simplified model. Further studies are needed to achieve a balance between high performance and reduced complexity of the model.

Abstract:Current study surveyed 70 sampling stations through four cruises in the adjacent seawater near Tangshan between 2006 and 2015. The contents and inter-annual variation characteristics of seven heavy metals: Hg, Cu, Pb, Cd, Cr, Zn and As in the surface sediment were analyzed. The single-factor exceeding rate, geo-accumulation index () and the comprehensive potential ecological risk index (RI) were used to evaluate the pollution degree. The results showed that the overall levels of the heavy metal contents in the surface sediment were low in the survey areas. Specifically, Cu and As contents in several stations exceeded the first class standard value, whereas the contents of other heavy metals accorded with the first class standard. The inter-annual content variations of the surveyed heavy metal were fluctuant slightly between 2006 and 2015. The contents of Cu, Pb, Cd, and Zn were relatively stable throughout the years, but the inter-annual variation of Hg and Cr increased initially followed by decrease. There was no significant change in As contents between the years. The evaluation results showed that the multi-year average of for the seven heavy metals was Hg>Cd>Pb>Cu>As>Cr>Zn, while the potential ecological risk assessment results showed that the multi-year average of the potential ecological risk factor for the seven heavy metals was Hg>Cd>As>Pb>Cu>Cr>Zn. Hg was the main pollution factor in surface sediments in the adjacent sea water of Tangshan based on the consistent results from two pollution evaluation methods, indicating that it has the highest ecological risk level. The inter-annual variations of RI ranged from 44.26 to 153.18 and the inter-annual average value was 89.92, belonging to the low ecological risk level. The trend of the inter-annual variations of RI was increasing and then decreasing, suggesting that the heavy metal pollution in surface sediments has been reduced in recent years.

Abstract:Patinopecten yessoensis is a cold-water bivalve commonly found in northwestern Pacific area including northern Japan, the Far East of Russian and the northern Korean Peninsula. It was introduced to China from the Mutsu Bay (Aomori Prefecture, Japan) and was aqua-farmed in the coast of the Bohai Sea and the north of the Yellow Sea in 1982. In this study, we applied stable isotope tracer technique on samples from different geographical populations of P. yessoensis to characterize their ecological adaptability in the northern Yellow Sea of China. Samples were collected from the bottom sowing population from Shandong Changdao, Liaoning Haiyangdao and Zhangzidao, the artificial population of Zhangzidao-Red, the natural colonized population in Liaoning Lvshun and the original natural population in the Aomori Mutsu Bay of Japan. The results showed that the δ13C value of the adductor muscle and the visceral mass of P. yessoensis were -20.47‰±0.66‰ and -24.13‰±2.41‰ respectively; the δ15N values of the two tissues were 8.52‰±1.32‰ and 7.26‰±0.49‰ respectively. Analysis of variance (ANOVA) indicated significant variances in δ13C and δ15N of the visceral mass and the adductor muscle among different populations; the scatter plot of 1δ13C versus δ15N for the two tissues across all samples showed apparent difference between the Chinese and Japanese populations. Among the 8 populations in China, the value of δ13C versus δ15N of the Changdao population was lower than those of other groups. These results indicated that significant divergences emerged between the Chinese and Japanese populations of P. yessoensis, as well as between different populations in China. This phenomenon could be explained by the ecological adaptability developed during the settlement in different environments. Our study helped understand the ecological adaptation process of the scallop P. yessoensis in the Yellow Sea, and provided useful information for promoting the economic benefits and sustainable development of the scallop culture industry in China.

Abstract:Breeding new varieties is a main approach to prevent the genetic degradation and improve traits for the purpose of economic benefits. Quantitative genetics evaluation is one of the main methods used in Japanese flounder (Paralichthys olivaceus) breeding. By using the core populations from the established breeding program, this research evaluated three growth related traits (body weight, total length, and body width) among 42 newly-generated families on 140 d, 170 d, 200 d (before mixing pool) and 380 d (after mixing pool). The quantitative genetic analysis was carried out using MINQUE, REML and BLUP. The results showed that the variation coefficient of growth traits ranged between 10.56% and 38.62% at different day age. Among three growth traits, the variation coefficient was the largest in body weight, and was relatively small in total length and body width. The variation coefficient of different traits decreased with the increase of day age. The additive variance component ratio for the three traits was between (0.13±0.01) and (0.29±0.06), whereas the random variance component ratio was between (0.71±0.06) and (0.87±0.01). By contrast, the narrow sense heritability ranged between (0.13±0.01) and (0.29±0.06) and the generalized heritability was from (0.15±0.01) to (0.54±0.06). All of the above genetic traits were highly significant (P<0.01). Comparison of genetic effects on three growth traits during different developmental stages revealed that four parental groups (F0990, F1005, KS, and F0719) showed significant positive effects, whereas six groups of parental groups (F0751, F0768, F0780, F09121, F0927, and RS) showed significant negative effect (P<0.01). By contrast, the rest of 32 parental samples showed no significant effect. The correlation coefficient was between 0.82 and 0.96 in phenotype and between 0.72 and 0.97 in genotype. The phenotypic and genetic correlation coefficient of different day ages were maintained at a relatively stable level. Breeding value of the 380-day age was estimated using the BLUP method. The results showed that breeding value of the parental fish body weight was between 14.63 and 100.05. Among all the families, the highest breeding values for body weight were found in F1005-8, F09119-11, F09125-4, F0915-57, F09104-12, F1264, F0908-38, F0927-20, F1005-53, F0990-6, F09125-7, F0751-14 and F1005-42, average between 20.87 and 35.60. The families with the highest average weight breeding value were F1416, F1428, F1442, F1418, F1427, F1408，F1402, F1412 and F1446. Compared with the method of phenotypic selection, the breeding values obtained from growth-based selection were better for improving growth traits since the selection efficiency was up to 81.91%. In this study, parents and families with superior growth traits were selected to improve the genetic quality and new varieties of P. olivaceus. Meanwhile, it provides an important theoretical basis for the development of P. olivaceus breeding program.

Abstract:Myosin heavy chain (MYH) is the main component of the skeletal muscle thick filament. The expression levels of MYHs have significant influences on the formation of thick filaments and muscle development. To understand the roles of MYHs on growth during the early development of mandarin fish (Siniperca chuatsi), the differentially expressed unigenes from the transcriptome sequencing data of the fast and slow growing fish were analyzed, and two highly expressed unigenes of MYH were screened. The full length cDNAs of these two MYH-7s, MYH-7a and MYH-7b were obtained by RACE method; they were 6070 bp and 5896 bp in length, with coding regions of 5820 bp and 5745 bp, respectively. Sequencing analysis showed that both MYH-7s contain the conserved regions such as Loop1 and Loop2, and ATP binding sites. The evolutionary tree clustering analysis showed that both MYH-7s genes belong to the slow muscle type MYH. The expression patterns of the two MYH-7s in different tissues were detected by qRT-PCR. The highest expression of MYH-7a was found in heart muscle while MYH-7b was highly expressed in red muscle. The expression levels of both MYH-7a and MYH-7b increased gradually in accordance with the embryonic development stages. The expression levels of the MYH-7s in the fast growing group were significantly higher than in the slow growing groups in 15, 30-day post hatching (dph), while both genes showed lower expression levels in fast growing group than in slow growing group in 60 dph. The different expression levels in tissues and early developmental stages of these two genes in the fast and slow growing groups suggested that they play important roles in the embryonic and early development of S. chuatsi.

Abstract:There are two kinds of male in the breeding populations of the half-smooth tongue sole Cynoglossus semilaevis, namely natural males (ZZ) and neomales (ZW). It is of great importance to study their phenotypic and genetic characteristics since the knowledge can improve its breeding techniques and seed cultivation. In this study, 15 phenotypic traits were measured between mature ZZ males and ZW males (sex-inverted males) of C. semilaevis. Analysis showed that highly significant differences between ZZ males and ZW males existed in seven traits, including body weight, full length, body length, body width, head length, eye spacing and eye diameter, with the growth rate of ZW males being higher than that of ZZ males. Furthermore, comparative analysis of the population genetic structures was processed among maternal half-sib families between ZZ and ZW males by using 50 pairs of specific microsatellite markers. Results showed that 22 out of 50 pairs of PCR amplified markers were polymorphic. Among four half-sib families, 232 alleles were identified with relatively higher mean allelic number (Na) and mean expected heterozygosity (He) compared to mean effective allelic number (Ne) and observed heterozygosity (Ho) respectively. In addition, the average polymorphism information content (PIC) was 0.4566, 0.4254, 0.5069, 0.4795 among four half-sib families and there were 3, 3, 4 and 5 loci significantly deviated from Hardy-Weinberg expected genotypic frequencies. The Nei´s genetic distance among the four families was 0.2101–0.5840, and the genetic similarity was 0.5577–0.8105. The current study indicated that there were significant differences among growth traits between ZZ and ZW males of C. semilaevis. Between the maternal half-sib families, there was relatively high genetic similarity, low genetic distance and similar genetic diversity index.

Abstract:CD59 is a glycophosphatidylinositol (GPI)-linked protein, identified as a regulator of the terminal pathway of complement membrane system. This study reported the gene expression patterns of CD59 of Japanese flounder (Paralichthys olivaceus) before and after challenged with Vibrio anguillarum. The results implied that few expression of CD59 was detected in all tissues before challenged with V. anguillarum. After being challenged, the expression levels of CD59 were significantly higher than the control group on different levels. And the most abundant expression was identified in kidney. Meanwhile the CD59 gene was inserted into the pET-32a to construct prokaryotic expression plasmid. The recombinant plasmid expressed in Escherichia coli BL21 (DE3) cells through isopropyl-β-thiogalacto- pyranoside (IPTG) induction and approximate 29 kDa recombinant protein of pET-32a-CD59 was obtained. Through the analysis of ultra-sonication on recombinant expression strains, the recombinant pET-32a-CD59 was identified in precipitate. It implied that the recombinant pET-32a-CD59 existed as an inclusion body. Then the recombinant protein was purified and concentrated by Ni-chelating affinity chromatography and ultrafiltration. SDS-PAGE analysis revealed a single band implying successful purification. Western blotting analysis showed that the recombinant protein could be specifically combined with anti-His monoclonal antibody. So the expressed protein was confirmed as the target protein. The recombinant protein was refolded by dialysis because of its incapability in biological activity. There was no precipitate or separation after refolding, which suggested that the operation of refolding by dialysis on recombinant protein was successful. After purification and refolding, the antimicrobial activity of the recombinant CD59 was demonstrated in Japanese flounder. Four pathogens were chosen for the experiment including Aeromonas hydrophila, V. anguillarum, Staphylococcus aureus and E. coli. The results revealed that recombinant Japanese flounder CD59 displayed antimicrobial ability through the inhibition on the growth of A. hydrophila. This study explored the immune regulation mechanism in Japanese flounder and provided molecular theoretical basis for improving the efficiency of breeding in Japanese flounder.

Abstract:The development of the digestive system of Centropristis striata and its associated glands were examined histologically from the first day (first day post-hatch, dph) until 34th day after hatching. Sampling was performed daily between 1 dph and 15 dph with sampling size around 30 fish. After that, sampling frequency was reduced to once every other day until 34 dph with sampling size of 15 fish. The newly-hatched larvae were reared in tanks with continuous supply of oxygen. Meanwhile salinity was about 30–32 and water temperature was maintained at (24±1)℃. It was found that the yolk sac of newly-hatched larvae was relatively large, while the digestive tract was a closed straight tube with a smooth lumen dorsally attached to the yolk sac. The mouth and anus formed and opened on the 3 dph, and followed by a 4-day period of mixed endogenous-exogenous feeding. Most of the yolk sac reserves were consumed rapidly; during this period, prey capture began and the digestive system continued to develop. Specifically, the digestive tract of larvae was differentiated into esophagus, stomach and intestine, and the digestive glands (1iver, gall-bladder and pancreas) began to form. Yolk sac and oil globule almost disappeared at 7 dph, and the nutritional requirements of the larvae has gradually made the transition from endogenous to exogenous. Mucous cells could be found in the epithelia of the oesophagus at 11dph, and the number of cells kept increasing during the larval development. Stomach was gradually differentiated into cardiacus, gastric body and pyloricus. At the same time, sinusoid and central veins of liver were clearly distinguishable. Gastric gland formed at 20 dph, which indicated the beginning of exogenous protein digestion. Increased wrinkles in the mucous layer and thicker muscles were detected in the intestine. Meanwhile, pancreas was abundant in zymogen granules spreading on the surrounding areas of the intestine. The structures and functions of digestive tract and digestive glands gradually became full-functional. It was concluded that C. striata larvae should be supplied with adequate and palatable food when they enter exogenous feeding period, and domesticated to feed formula feed after 20 dph. The findings on the development of the digestive system lead to a better understanding of the ontogeny of C. striata, which would be useful for improving the larval rearing techniques of this promising Centropristis species and therefore diversifying the freshwater aquaculture.

Abstract:An 8-week feeding trial was conducted to investigate the effects of different types of protein hydrolysate in high plant protein diets on the lipid accumulation of juvenile Japanese seabass Lateolabrax japonicus (initial body weight 31.99 g). Four experimental diets were formulated. The control diet contained 30% fishmeal. Different types of protein hydrolysate, i.e., pig blood protein hydrolysate, soy protein hydrolysate and yeast protein hydrolysate was separately added to the basal diet to replace 10% fishmeal to formulate three experimental diets. The feeding trail was conducted in sea floating net cages. Each diet was assigned to triplicate groups of 30 fish. The results showed that compared to the control group, the supplementation of protein hydrolysates from pig blood, soybean meal and yeast did not significantly influence most indices of lipid accumulation in L. japonicus. However, compared to the control group, the yeast protein hydrolysate significantly reduced the concentrations of triglyceride, cholesterol, and low density lipoprotein cholesterol in serum (P<0.05). The fatty acid analysis showed that compared to the control group, the yeast protein hydrolysate significantly increased the C20:1n-9 content in muscle, while the soy protein hydrolysate and yeast protein hydrolysate significantly reduced the C22:5n-3 content in muscle (P<0.05). Compared to the control group, the yeast protein hydrolysate significantly reduced the C20:5n-3 content in serum (P<0.05). In adipose tissue, compared to the control group, the protein hydrolysates from pig blood, soy, and yeast significantly increased the contents of saturated fatty acids, ≤18C monounsaturated fatty acids, linoleic acid and linolenic acid, but significantly reduced the contents of C20:1n-9、C20:4n-6、C22:5n-3 and C22:6n-3 (P<0.01). These results suggested that in high plant protein diets the supplementation of protein hydrolysates from pig blood, soy, and yeast did not modulate the lipid accumulation in whole body, liver, muscle and gut of juvenile L. japonicus, but the yeast protein hydrolysate significantly reduced the concentrations of triglyceride, cholesterol, and low density lipoprotein cholesterol in serum. The modulation of fish fatty acid profiles by protein hydrolysates varied depending upon the type of protein hydrolysate and the type of fish tissue. This is the first study comprehensively investigating the effects of different types of protein hydrolysate on the lipid accumulation in aquaculture species, and the results were indicative for the relevant studies in the future.

Abstract:This study was conducted to investigate the effects of four protein hydrolysates for replacing fish meal protein of basal diets on growth performance and non-specific immunity of Japanese seabass (Lateolabrax japonicus). The control group included fish meal (FM) as the single protein source. The experimental groups were replaced 10% of fish meal protein with four protein hydrolysates, including fish protein hydrolysate (FPH), pig blood protein hydrolysate (PBPH), soy protein hydrolysate (SPH) and yeast protein hydrolysate (YPH). Five diets were formulated to be isonitrogenous and isolipidic. All diets were fed in triplicate groups of L. japonicus (initial weight 31.99 g) for 8 weeks. Results indicated that the final body weight and specific growth rate (SGR) were significantly improved when fish were fed the FM and FPH groups compared with the PBPH, SPH, and YPH groups (P<0.05), but there was no significant effect on survival rate and feed intake among all groups (P>0.05). There was no difference in feed efficiency (FE) among the FPH, FM, PBPH, and YPH groups (P>0.05), while the FPH group was significantly higher than the SPH group (P<0.05). The FPH group had significantly higher protein efficiency ratio (PER) than other dietary treatments (P<0.05). The FPH, FM and YPH groups showed significantly higher protein productive value (PPV) than that of PBPH and SPH groups (P<0.05), while PPV in the FPH, FM and YPH groups were not significantly different (P>0.05). AKP, ACP, SOD, LZM activities and T-AOC in liver and serum of the FPH and YPH groups were significantly higher than other groups (P<0.05). The FPH group showed significantly highest crude protein of fish body in all dietary treatments, but there were no significant different between the FPH, FM and YPH groups (P>0.05). In conclusion, FPH had the best effects on growth and non-specific immunity of L. japonicus compared with other diets, followed by YPH, PBPH and SPH.

Abstract:GWT is an ingredient blend mixing vital wheat gluten, wheat flour and taurine with the ratio of 77.5%, 20.5%, and 2.0%, respectively. This study was conducted to evaluate GWT as a protein source to compare with low-temperature dried fish meal (LT-FM) and soy protein concentrate (SPC) in extruded practical diet to feed to juvenile giant croaker (Nibea japonica) and black sea bream (Sparus macrocephalus). Plasma biochemical indices and liver anti-oxidative indices of the two fish species were tested as fish health indicators in this study. Eight diets had been formulated, including a control diet with LT-FM (20%) and SPC (21.4%), six diets with decreasing percentages of LT-FM or SPC replaced by GWT (replacing levels were 33.3%, 66.7% and 100%, respectively, on protein basis) and one diet with both 50% of LT-FM and SPC was replaced by GWT. Each diet was fed to triplicate tanks of giant croaker with an initial weight of (12.83±0.91) g (26 fish per tank) and black sea bream with an initial weight of (15.40±0.02) g (35 fish per tank). All fish were reared in a sea-water flow-through system for 59 days. The results showed that there was no significant effect of replacing LT-FM by GWT on total protein (TP) content, plasma triglyceride (TC), and MDA content and liver SOD activity in giant croaker (P>0.05). However, total cholesterol (TG) and glucose (GLU) contents in plasma were significantly decreased (P<0.05), whereas the plasma SOD activity of juvenile giant croaker increased significantly (P<0.05) when 66.7% and 100% of dietary LT-FM was replaced by GWT. Partial or total replacement of LT-FM or SPC by GWT had no significant effect on plasma levels of TP, TG, TC and GLU, liver MDA content and plasma SOD activity of black sea bream (P>0.05). In contrast, significant increase of liver SOD activity was found when total LT-FM, 33.3% and 66.7% of SPC, and 50% of LT-FM and SPC in both were replaced by GWT in diet of black sea bream. To conclude, giant croaker was more sensitive than black sea bream to the significant reduction of LT-FM in the low fish meal based diet, with the changes of plasma biochemical indices and liver anti-oxidative indices. Highlights of the present study: firstly, it is the first study using GWT as main protein source in extruded diets for giant croaker and black sea bream; secondly, a low fishmeal (20%) containing extruded practical diet was used as the control.

Abstract:Long-chain polyunsaturated fatty acids (LC-PUFA) are essential fatty acids (EFA) and have important roles in growth, development and physiological function of fish. Fish oil is characterized by high content of n-3 LC-PUFA, but harvesting fish oil is controversial due to limited wild fish stock and increased production cost. Silver pomfret (Pampus argenteus) is an important economic species in coastal areas of China. There are many problems in the technological development of artificial breeding and aquaculture for silver pomfret. For the purpose of researching and developing artificial feed, reducing feed cost, improving fish health and survival rate, in this study, 4 experimental dietary groups were designed for the feeding trial: 100% fish oil (FO), 70% fish oil and 30% soybean oil (FSO), 30% fish oil and 70% soybean oil (SFO), and 100% soybean oil (SO). To explore the effect of substitution of dietary fish oil by soybean oil on silver pomfret, serum lysozyme (LZM) activity and tissue antioxidant capacity were measured. The results suggested that serum LZM activities were the highest in SFO group, and the lowest in SO group, although no significant difference was detected (P>0.05). Muscle malondialdehyde (MDA) contents of FSO and SFO group were significantly higher than that of FO and SO group (P<0.05). Liver MDA contents in FO and FSO groups were significantly higher than that of any other groups (P<0.05). The highest superoxide dismutase (SOD) and catalase (CAT) activities of serum and muscle were in SFO group. By contrast, SO group had lower SOD and CAT activities than any other groups. The liver SOD and CAT activities were found significantly higher in FO group than other groups (P<0.05). T-AOC of muscle and liver in FO and FSO were higher than other groups, and group SO had the highest serum T-AOC. This experiment suggested that replacing dietary fish oil by soybean oil for 30% or 70% can promote immune and antioxidant function of juvenile silver pomfret. But replacing dietary fish oil by 100% soybean oil had negative effect on immunity and growth of silver pomfret.

Abstract:In the present study, we explored the effect of poly-β-hydroxybutyrate (PHB) on the performance of the immune system in Fenneropenaeus chinensis. This study used single factor concentration gradient method. F. chinensis were fed with baits containing PHB at different concentrations (0, 0.5%, 1.0%, 2.5% and 5.0%). After six weeks, the mortality and relative immune protective rate of each group was calculated. Moreover, enzyme activities of nonspecific immunity, including the total antioxidant capacity (T-AOC), the enzyme activities of acid phosphatase (ACP), peroxidase (POD) and catalase (CAT), and the content of malondialdehyde (MDA) in muscle, hepatopancreas and serum were analyzed and compared between groups on a weekly basis. The results showed that the PHB had a significant effect on immune performance. With the increase of PHB concentration, the relative immune protective rates in the experimental groups were elevated initially followed by a decline. The group of E1.0 had the highest relative immune protective rate compared with other groups (P<0.05). The trend of the enzyme activities were similar to the changes of the relative immune protective rates. On the spatial distribution, the enzyme activities of T-AOC, POD, CAT in serum and ACP in hepatopancreas were the highest. On the temporary distribution, the total enzyme activities reached the highest level after 2–3 weeks of treatments. Among different groups, the groups of E1.0 and E2.5 in serum and E1.0 in hepatopancreas had significant different T-AOC from other groups (P<0.05). The ACP activities in serum in the groups of E1.0 and E2.5, and E1.0 in the hepatopancreas were significantly different from other groups (P<0.05). During the high level expression period of CAT, serum CAT in E0.5, E1.0, E2.5 groups, hepatopancreas CAT in E 0.5, E1.0, E10.0 groups were significantly different from other groups (P<0.05). The serum and hepatopancreas POD activities in E0.5, E1.0, E2.5 groups were significantly different from other groups (P<0.05). The SOD activities in serum and hepatopancreas in group E1.0 were significantly different from other groups (P<0.05). And the content of MDA in serum in E1.0 group and that in hepatopancreas in group E0.5 and E1.0 were significantly different from other groups. Based on the results, it is concluded that the PHB can improve the immunity of F. chinensis. Furthermore, 1.0% PHB inclusion in feed had the best effect and the total enzyme activities reached the highest level after 2–3 weeks of treatment.

Abstract:In order to analyze genetic effect of the ridgetail white prawn (Exopalaemon carinicauda) backcross families, 25 microsatellite loci were selected to detect the genetic characteristics of three backcross family A, B, C, generated by our laboratory. The results showed that among A, B and C families, the average number of alleles (Na) were 2.21, 2.18 and 2.09, the observed heterozygosity (Ho) were 0.3805, 0.3703 and 0.2489, the expected heterozygosity (He) were 0.3629, 0.3998 and 0.2503 and the polymorphism information content (PIC) were 0.3257, 0.3391 and 0.2585 respectively. Compared to the wild and hybrid population, the genetic diversities of the backcross families were greatly reduced which belonged to moderate or low polymorphism. 62 alleles were obtained in the three backcross families in total using 25 loci. Specifically, 56, 54 and 51 alleles were obtained in A, B and C backcross families, respectively. In addition, there were 39 common alleles in the three backcross families. Six loci have homozygosity in all backcross families (ECL10 in A, B and C families). The average homozygous rate was 53.86%, 56.53% and 63.33% respectively, which showed that the purification rate of gene was rapid and the backcross is suitable for selective breeding in E. carinicauda.

Abstract:Shrimp aquaculture, as one of the main components of the aquaculture industry, has produced enormous economic benefits and social benefits. But the sustainable development of the shrimp aquaculture has been affected and restricted by the discharge of the waste and the frequent occurrence of diseases. Bio-floc technology by adding brown sugar, molasses and other carbon source in breed pool to promote heterotrophic microbial growth and reproduction to control the water pollution, shows great prospects for development in shrimp culture. In this study, we established a method of simplified fermentation that used a strain of Bacillus megaterium with the activity of denitrification. And we used prawn feed and brown sugar as the medium. Results showed that the best ratio of medium was prawn feed 3 g/L, brown sugar 6 g/L and inoculation volume 1×108 CFU/ml through the orthogonal experiment. And the other conditions were optimized by single experiment. We got the fermentation products with the viable cell number was 1.16×1010 CFU/ml that fermented with 31℃, 40% liquid volume and 24 hours. Then we fed the Litopenaeus vannamei through the bio-floc technology by using the fermentation products. The result showed that the volume of bio-floc was formed more quickly in dominated bio-floc group (added bacillus and sugar) than bio-floc group (just added sugar) and control (traditional culture pattern) (P<0.05), and the overall concentration level of nitrite-N in dominated bio-floc group was much lower than the other groups (P<0.05). At the end of the culture, the highest of the average body length and weight in prawns was obtained in dominated bio-floc group, and Bio-floc group and control were significantly lower (P<0.05). This study established a simplified and feasible method for the fermentation of probiotic bacteria. And the results showed the effectiveness of the bio-floc technology in prawn culture is verified by the additiong probiotics.

Abstract:The influence of temperature fluctuation on physiological parameters and immune activity of three different sized scallop (Patinopecten yessoensis) was studied in this work. Temperature fluctuated in the range of 15-10-15℃, with increasing or decreasing 5℃ per 2 h. Temperature was totally fluctuated four times in the experiment, which named as B1, B2, B3 and B4. Results showed that the mortality rates of big, medium and small sized P. yessoensis were 4%, 6% and 6%, respectively. The mortality rate of big sized P. yessoensis was relatively lower, and the mortality only appeared in B1 and B2. Oxygen consumption rates of three different sized scallop were lower at B1 than the initial value of B0. With temperature fluctuation, oxygen consumption rates gradually increased and then were higher than B0 in the end. Ammonium excretion rates (AER) decreased with temperature fluctuation, except B1 of the small sized scallop. For the big sized scallop, the AER in B3 was significantly lower than that in B0 (P<0.05), where for medium sized scallop, AER in B4 was significantly lower than that in B0 (P<0.05). Comparing with physiological parameters, the immune activities were more sensitive to temperature fluctuation. Activities of superoxide dismutase (SOD) and catalase (CAT) decreased significantly as the temperature fluctuated 1 or 2 times (ANOVA, P<0.05). In conclusion, the temperature fluctuation in optimum range had impact on the physiological parameters and immune activities of scallop P. yessoensis to a certain extent.

Abstract:In order to provide reliable theoretical basis for high-efficiency utilization of Saccharina japonica germplasm resources in its breeding practice, the information of genetic diversity and structure of three new edible S. japonica cultivars (including “Haitian No.1”, “Haitian No.2” and “Haitian No.3”) and “Huangguan No.1” were explored in this study. We selected eight pairs of primers for the simple sequence repeat (SSR) molecular marker technique to test the genetic diversity of 120 samples of the four S. japonica cultivars. From 20 pairs of SSR primers published online, eight pairs were identified because of their good amplification patterns on the majority of 120 DNA templates. The results showed that 28 polymorphic alleles and 9 specific alleles were detected by eight SSR markers, and the average alleles per primers detected were about 4.6250. The Nei¢s gene diversity (H) and the Shannon index (I) of the four S. japonica cultivars were 0.3809 and 0.6702, respectively. It indicated that the genetic diversity of the four S.japonica was relatively low and four species were genetically related. Among them, the Nei¢s gene diversity (H), Shannon¢s information index (I) and the number of polymorphic alleles were the highest in “Haitian No.1”. This indicated that the genetic diversity of S. japonica of “Haitian No.1” was higher than other three S. japonica cultivars. The levels of genetic diversity of “Haitian No.2”、“Haitian No.3” and “Huangguan No.1” decreased successively. Cluster analysis demonstrated that the relationship between “Haitian No.1” and “Haitian No.3” was close, but the relationship between “Haitian No.1” and “Huangguan No.1” was not. AMOVA analysis showed that about 92.06% of the variation was derived from individuals of the four S. japonica cultivars, and 7.94% of the variation was derived among the four S. japonica cultivars. The genetic diversity of “Huangguan No.1” was the lowest, suggesting the need of strengthening the biodiversity protection; the highest genetic diversity was the “Haitian No.1”, and could be used for developing new cultivars.

Abstract:Bacillus firmus strain PC024, isolated from Chinese shrimp (Fenneropenaeus chinensis) rearing environment, has demonstrated its ability to increase shrimp resistance to white spot syndrome virus (WSSV) infection and to ferment dietary soybean meal. The purpose of this current study is to optimize the processing conditions for the fermentation of soybean meal using B. firmus. Considering single factor trials, results indicated that the optimum bacterial concentration for inoculation, water ratio, fermentation time and temperature were 2×106 CFU/g, 1:0.8, 90 h, and 37℃, respectively. However, on the basis of univariate tests, the response surface methodology (RSM) was applied to optimize four fermentation factors when they were combined, including temperature, time, water ratio, and amount of bacteria required for inoculation. The optimum fermentation conditions were as follow: fermentation temperature, 39.0℃; fermentation time, 100 h 18 min; water ratio, 1:0.96; and amount of bacteria for inoculation, 3.84×106 CFU/g. Under the optimized conditions, the bacterial concentration reached 1.23×1010 CFU/g after fermentation and it was 5.13% deviated from the predicted value using the RSM model. The quality of soybean meal improved significantly after fermentation. Firstly, the yield of the fermented soybean meal reached (93.89±0.01)%. Secondly, after fermentation, the soluble protein content in the soybean meal increased from (39.16±0.01)% to (58.80±4.54)%, and crude protein content in the soybean meal increased from 50.71% to 55.03%. Finally, the total amount of amino acids (15 types) had 132.30% increase compared to the original content; the most significant increases in the proportion amino acids were arginine (168.60%), lysine (157.20%), serine (152.50%), threonine (139.04%) and glycine (138.40%). It showed that the protein molecules were effectively degraded in SDS-PAGE. This study provides theoretical support for the application of probiotics in shrimp plant-basis diet and disease control in aquatic animals.

Abstract:In intensive culture system, accumulation of ammonia nitrogen, which is the principal end-product of nitrogenous metabolism, has deleterious effects on aquatic animals. However, there is little information about the effects of ammonia nitrogen on the immunity function of sea cucumber Apostichopus japonicus. This study was conducted to evaluate the effects of ammonia nitrogen stress (concentration gradient: 0, 2, 4, 6, 8, 10 mg/L) on the activities of lysozyme(LSZ), catalase(CAT), superoxide dismutase (SOD) of different-sized sea cucumber with initial body weight of (6.05±0.44) g, (14.68±1.76) g and (25.64±3.27) g. The results were as follow: When the ammonia-N level was 8 mg/L, mortality occurred in three-sized sea cucumber on the 10th day; When the ammonia-N level was 10 mg/L, the medium-sized sea cucumber had the lowest survival rate of 86.7%. When the ammonia-N level was 2 mg/L and 4 mg/L, on the 4th day, three non-specific immune enzymatic activities of three-sized sea cucumber increased, significantly different with the control group (P<0.05). There were no significant differences in enzymatic activities between the 4th, 7th, 10th and 13th day. While the ammonia-N level was 6 mg/L, 8 mg/L, 10 mg/L, three non-specific immune enzymatic activities reached the maximum value on the 7th day, significantly different with the control group (P<0.05). Under the same ammonia nitrogen stress level, the activities of three non-specific immune enzymes of three-sized sea cucumber were different. The sensitivity order of three-sized sea cucumber under ammonia nitrogen stress was: Medium-sized>Big-sized>Small-sized (P>0.05). These results indicated that ammonia nitrogen stress can affect the survival rate and immunity function of sea cucumbers, which will provide the theoretical basis for the mechanism of ammonia stress on sea cucumbers.

Abstract:From December 2014 to December 2015, the flora population structural analysis and beneficial bacteria isolation and screening were carried out in large water ponds of a sea cucumber (Apostichopus japonicus) farm. Sixty-six bacterial strains were isolated from the water body and pond sediment. Two bacterial strains Pseudoalteromonas nigrifaciens and Vibrio splendidus associated with the “skin ulcerative syndrome” disease were used as indicators in the antagonistic effect trials. Based on the bacterial extracellular amylase, protease activities and challenge tests, one potential probiotic bacterium YQ-2 was obtained. The results showed that this strain had good ability to inhibit the proliferation of P. nigrifaciens and V. splendidus, evident by the diameter of inhibition zones, 22 mm and 24 mm, respectively; while the diameter of amylase and protease hydrolysis was 22 mm and 36 mm; In addition, the challenge tests revealed that the strain YQ-2 was safe to sea cucumber when it was challenged by bathing the bacterial strain at the concentration of 108 CFU/ml, or fed the food inoculated with the bacterial cells at the concentration of 108 CFU/g once daily for one month. The present study also found that among the sea cucumbers fed the food containing 108 CFU/g bacterial cells, the body weight increased significantly compared with the control group, with the relative growth rate as high as 39.31%. Homogenous analysis using 16S rDNA sequences indicated that the strain YQ-2 had 99% similarity with the Bacillus subtilis strain KLP2015. Thus, the strain YQ-2 was tentatively identified as Bacillus subtilis. The number of B. subtilis in the rearing water was counted in all four seasons and the year-round concentration was about 140–280 CFU/ml, which was higher than other strains. Meanwhile the dominance of YQ-2 was 4.2%, 3.5%, 2.6%, and 4.6% corresponding to Spring, Summer, Autumn and Winter. Furthermore, the dominance of YQ-2 was higher in Spring and Winter compared to Summer and Autumn. Nevertheless, the strain YQ-2 exhibited a strong antibacterial effect on the pathogens P. nigrifaciens and V. splendidus which were the main pathogenic agents causing the “skin ulcer syndrome” disease. Hence, the strain YQ-2 has great application for farming A. japonicus in large water ponds.

Abstract:The cultured juvenile sea cucumber (Apostichopus japonicus) sufferred from "enteritis disease" in a breeding farm in Penglai in March 2014. In this study, the clinical symptoms and histopathology of enteritis disease were investigated. To provide reliable means to control this disease, the pathogen was isolated and identified, and its sensitivity to different antibiotics were tested. The effects of different therapeutic methods (oral, bath, oral with bath) were evaluated using three indices, including the ratio of viscera to body wall (RVBW), weight gain rate (WGR) and specific growth rate (SGR). The results showed that the sea cucumber infected with the enteritis disease displayed dark body color, poor feeding rate, weak motility and adhesion ability. The ingested food in the intestine of sick sea cucumber was discontinuous, and large amount of yellow-white mucus was found in the intestine. The intestinal wall was brittle, poor toughness and easy to fracture. A large amount of bacteria was detected in the intestinal tract under the microscope. The histopathological analysis on infected sea cucumbers revealed the scattered intestinal villus, dispersed mucous layer, scattered connective tissue and obviously separated muscle layers. One dominant bacterium (In-1) was isolated from the infected sea cucumber intestine and was confirmed as the causative pathogen using the artificial infection experiment. The bacteriological analysis indicated that the deteriorated feed (algae powder) was most likely the origin of the pathogen. The pathogen was identified as Vibrio harveyi using morphology observation, physiological and biochemical, and molecular methods (16S rDNA and gyrB). The strain was highly sensitive to 12 kinds of drugs such as ceftriaxone, azithromycin, doxycycline and florfenicol. The medication treatment using florfenicol with three different methods showed that medicated bath treatment was the most effective and achieved the best results of the WGR of (24.23±0.41)% and SGR of (0.77±0.01) %/d. The study provides the theoretical basis and technical reference for disease prevention and control in sea cucumber culture and its healthy breeding.