花鲈弧菌病病原菌（哈维氏弧菌）的分离与鉴定

1999年4－5月，山东省青岛市胶南海区网箱养殖场花鲈（Lateolabrax japonicus)幼鱼发生暴发性传染病，死亡率达50％。从具有明显症状的病鱼的病灶组织分离到1株优势菌SF－1，经人工感染和从人工感染发病的花鲈再分离的SF－3菌株的再感染试验结果表明，所分离的菌株为此次花钙烂层病的致病菌，经形态、生理生化等64项特征指标鉴定，SF－1和SF－3均为哈维氏弧菌（Vibrio harveyi)。药敏试验结果表明，头孢噻肟，头孢三嗪，头孢孟多，呋喃妥因，氯霉素,氧哌嗪青霉素，磺胺类，复方磺胺、三甲氧苄氨嘧啶，多粘菌素E等10种药物对该菌株有明显的抑制作用。     

Effects of fish meal replacement by soybean meal with supplementation of functional compound additives on intestinal morphology and microbiome of Japanese seabass (Lateolabrax japonicus)

A 15‐week trial was carried out to estimate the effects of functional compound additives (FCA) on intestinal morphology and microbiome in Japanese seabass, Lateolabrax japonicus, fed diets with soybean meal (SBM) partially replacing fish meal (FM). The formulation of FCA was the mixture of antioxidant, immunopotentiator and Mintrex^® trace elements. Four isonitrogenous (45%) and isolipidic (11%) diets, including FM42 (the control group without FCA), FM35 (15% FM protein substitution level with FCA), FM21 (50% FM protein substitution level with FCA) and FM21‐N (50% FM protein substitution level without FCA) were formulated. Quadruplicate groups of seabass (initial average weight 125.65 ± 0.60 g) were randomly handfed each diet twice daily in seawater floating cage. No significant differences were found in microvillus height (HMV) and muscular thickness (MT) of distal intestine among fish fed FM42, FM35 and FM21, whereas the HMV, MT and fold height (HF) in seabass fed FM42 were significantly higher than those of seabass fed FM21‐N (P < 0.05). Seabass fed FM21‐N had more obvious enteritis parameters such as tissue disruption, wider lamina propria and less mucosal fold than those in seabass fed FM21. High‐throughput sequencing technology of gut flora showed that Proteobacteria was the most dominant bacteria at phylum level among groups. There was no significant difference in relative abundance and composition of gut microflora among groups. In conclusion, supplementation of FCA might partially eliminate SBM‐induced enteritis, but the composition and relative abundance of intestinal microflora were not affected by dietary treatments.     

Molecular cloning and functional characterization of the fatty acid delta 6 desaturase (FAD6) gene in the sea cucumber Apostichopus japonicus

The sea cucumber (Apostichopus japonicus), an important echinodermata, had high value in nutrition and medicine for its rich collagen, sulphated polysaccharide, glycosides and polyunsaturated fatty acids (PUFA). The cDNA of the fatty acid desaturase gene in A. japonicus (AJFAD6) was cloned and was found to encode a desaturase with delta 6 FAD activity. Sequence analysis indicated that AJFAD6 included an open reading frame of 1392 bp, encoding 463 amino acids. AJFAD6 has all the conserved motifs found in other members of the FAD6 family, including an N‐terminal cytochrome b5 domain and three histidine‐rich regions. qRT‐PCR showed that AJFAD6 was expressed in all tissues tested during juvenile development and was mainly expressed in the respiratory tree at 150 days after adherence (150 days) and in the intestine at 100 days. Furthermore, AJFAD6 mRNA was also detected in the analysed adult tissues, with higher expression in the intestine and testis. Functional characterization of AJFAD6 in a recombinant yeast, Pichia pastoris, showed that AJFAD6 could catalyse exogenous linoleic acid (LA) and α‐linolenic acid (ALA) to produce γ‐linoleic acid (GLA) and stearidonic acid (STA), respectively, at conversion rates of 11.1% for LA to GLA and 3.4% for ALA to STA. Our results suggested that the biosynthetic pathway of PUFA existed in the sea cucumber, but endogenous production of eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid from either LA or ALA precursor appeared to be limited.     

Effects of dietary copper sources on growth,tissue copper accumulation and physiological responses of Japanese sea bass (Lateolabrax japonicus) (Cuvier, 1828) fed semipurified or practical diets

An 8‐week trial was conducted to compare the efficacy of a chelated copper (Cu) with the inorganic Cu (CuSO₄), in the diet of Japanese sea bass (Lateolabrax japonicus). Six diets were prepared based on two basal diets (semi‐purified or practical) at two Cu inclusion levels (0 and 4 mg kg^?1 from or chelated‐Cu), accordingly named as S0, SI4, SM4, P0, PI4 and PM4 respectively. Each diet was fed to quadruplicate groups of Japanese sea bass (initial weight: 65.0 ± 0.03 g) during the trial. Fish fed practical diets had higher feed intake, specific growth rate, protein efficiency rate (PER), liver Cu accumulation, liver Na⁺K⁺‐ATPase and plasma alkaline phosphatase, but lower hepatosomatic index than the groups fed semi‐purified diets (P < 0.05). Higher PER was exhibited by fish fed chelated‐Cu diets than that of fish fed CuSO₄ diets (P < 0.05). Although liver Cu accumulation was significantly higher in fish fed practical diets,, liver Cu content increased significantly with increasing dietary copper levels only in fish fed semipurified diets. Chelated‐Cu has higher efficiency than CuSO₄ when fed in practical diets. Poor growth performance of fish fed semipurified diets might induce underestimating the nutrient requirement of Japanese sea bass.     

Utilization of dietary carbohydrates by sea cucumber Apostichopus japonicus (Selenka) as indicated by carbon stable isotope analysis

Carbon stable isotopes were used as trophic markers to investigate the utilization of dietary terrestrial‐source carbohydrates by sea cucumber Apostichopus japonicus. Sea cucumbers were fed by five different types of diets with the ingredients containing Sargassum muticum either without starch or with one of the four starches including corn starch, sweet potato starch, tapioca starch and potato starch. After the 70‐day feeding trial, the carbon isotopic compositions of A. japonicus appeared to reflect those of corresponding dietary components. The average contribution of corn starch (22.0%) to the growth of A. japonicus was slightly higher than the expected contribution (20%). While the proportional contributions of sweet potato, tapioca and potato starches (6.0%, 7.0% and 4.0%, respectively) were all considerably lower relative to the expected contributions. These results indicated that A. japonicus could utilize corn starch more efficiently than sweet potato, tapioca or potato starch. Moreover, A. japonicus fed diet containing corn starch showed the highest specific growth rates which were significantly higher than those fed diets containing potato or tapioca starch. The results of the present study suggested that the corn starch could be used as dietary carbohydrate source in the artificial feeds for A. japonicus farming.     

The ratio between digestible protein and digestible energy affects accumulation and depuration of geosmin and 2-methylisoborneol (2-MIB) in Japanese seabass (Lateolabrax japonicus) raised in a recirculated aquaculture system

Actinobacteria and cyanobacteria accounted for less than 1% of total of bacteria in water in a recirculated aquaculture system (RAS) during a 15-week feeding trial with 0.11-g Japanese seabass. Resulting concentration of geosmin and 2-methylisoborneol (2-MIB) in RAS water was 169 and 45 ng L^?1, sufficient to produce strong off-flavour. The seabass were fed diets with 42%, 45% and 49% protein, and each protein level was supplemented with 15% or 18% lipid. Accumulation of off-flavours was independent of diet in fatty ventral tissue. Dietary protein significantly reduced off-flavours in lean, dorsal tissue. This was mainly rationalized by linear reduction in 2-MIB in response to increasing DP/DE and a strong, 2nd degree polynomial response in geosmin. The ratio between geosmin and 2-MIB was slightly higher at the beginning of a 10-day period with clean water and fasting, than what was observed throughout depuration. 2-MIB remained between 0.2 and 1 µg kg^?1 in dorsal tissue throughout depuration. Geosmin in ventral tissue ranged from 10 to more than 30 µg kg^?1at the termination of the feeding period and was reduced to a range from 6 to 20 µg kg^?1 by depuration.     

Molecular cloning of heat shock protein 60 from Marsupenaeus japonicus and its expression profiles at early developmental stages and response to heat stress

Water temperature is an essential environmental factor in aquaculture that affects many aspects of organism, and a rise in water temperature stresses aquatic animals. HSP60 is a major component of the chaperone system and plays an important role in stress response. In this study, the full‐length cDNA sequence of HSP60 from Marsupenaeus japonicus was cloned for the first time, and the tissue distributions and expression profiles of MjHSP60 at the early developmental stages of M. japonicus and under heat stress were verified by qRT‐PCR. The full‐length cDNA sequence of MjHSP60 was 2,303 bp with the deduced amino acid (AA) sequence of 579 AA. MjHSP60 was expressed in all eight tested tissues of M. japonicus and was mainly expressed in the hepatopancreas under normal condition. MjHSP60 was expressed in all of the early developmental stages examined from the fertilized egg to the post‐larval stage, and the expression level of MjHSP60 peaked at zoea II and reached a trough at the transition periods between each two stages (N and M1) and showed a relatively stable expression level at the post‐larval stage. The expression level of MjHSP60 was significantly up‐regulated in the gills, muscle, heart, hepatopancreas and stomach at 3 hr post‐heat‐stress and showed varied sensitivity to heat stress. The expression profile of MjHSP60 in the hepatopancreas and gill showed a time‐dependent manner post‐heat‐stress and peaked at 3 hr post‐heat‐stress. The present study provided a basis for further studies for elucidating the function of MjHSP60 in the heat stress response and the early developmental stages of M. japonicus.     

Microbial communities in sea cucumber (Apostichopus japonicus) culture pond and the effects of environmental factors

This study investigated microbial community composition as well as their correlation with environmental factors of Apostichopus japonicus culture ponds in northern China by 16S rRNA gene amplicon sequencing. The results showed that microbiota richness varied consistently with diversity in the pond ecosystem. Microbiota richness and diversity were highest in sediment, followed by gut of A. japonicus and water. The dominant bacterial phylum in the pond ecosystem is Proteobacteria. Gammaproteobacteriaeria and Flavobacteria are two dominant bacterial classes in the ecosystem. There is significant difference (p < 0.05) between dominant bacterial communities at the levels of order, family and genus. There is also remarkable regional difference (p < 0.05) between microbial community composition in the pond ecosystems. Specifically, microbial community composition in Changhai and Yingkou show a high similarity, so do those of Laoting and Rushan. According to the redundancy analysis of the microbial community composition and pond environmental factors, chemical oxygen demand is the dominant environmental factor determining microbial community composition in pond water; sulphide has the greatest influence on the microbial community composition in pond sediment; the rest of environmental factors have varied influence on microbial community composition in pond ecosystems.     

Effect of replacing dietary fish meal with silkworm (Bombyx mori L) caterpillar meal on growth and non‐specific immunity of sea cucumber Apostichopus japonicus (Selenka)

The effect of dietary substitution of silkworm (Bombyx mori L) meal (SM) for fishmeal (FM) on the growth performance and non‐specific immunity of sea cucumber (Apostichopus japonicus) (initial weight: 12.8 ± 0.16 g) was determined. Four isonitrogenous and isocaloric diets were formulated: Diet 1, which served as the control diet, contained 5% FM; Diet 2 contained 3.75% FM and 1.25% SM; Diet 3 contained 2.5% FM and 2.5% SM; and Diet 4 contained 5% SM. Other ingredients in each of the four diets were kept in the same proportion. After 8 weeks of feeding, the results showed that sea cucumbers fed Diet 2 had 18.7% increases in weight over those fed the control diet, but no significant difference was observed. No obvious difference in body wall composition was detected among the sea cucumbers fed the four different diets. Immunity analysis indicated that phagocytosis and serum alkaline phosphatase activity were not significantly (P > 0.05) affected when FM was partially or completely replaced with SM. Serum lysozyme activity of sea cucumbers fed Diet 4 showed a significant (P < 0.05) growth increase compared with those fed control diet. The results revealed that SM could be an effective substitute for FM in sea cucumber diet.     

Dietary docosahexaenoic acid to eicosapentaenoic acid (DHA/EPA) ratio influenced growth performance,immune response,stress resistance and tissue fatty acid composition of juvenile Japanese seabass,Lateolabrax japonicus (Cuvier)

A 10‐week feeding experiment was conducted to investigate the effects of dietary DHA/EPA ratio on juvenile Japanese seabass reared in sea floating cages. Six practical diets were formulated differing only in DHA/EPA ratio: 0.55 (Diet D/E0.55), 1.04 (D/E1.04), 1.53 (D/E1.53), 2.08 (D/E2.08), 2.44 (D/E2.44) and 2.93 (D/E2.93). All diets had the same contents of total n‐3 long‐chain polyunsaturated fatty acid (LC‐PUFA) and arachidonic acid (ARA). The results showed that the final weight and specific growth rate significantly increased with increasing dietary DHA/EPA ratio from 0.55 to 2.08 and thereafter declined. Activities of lysozyme and superoxide dismutase in serum in groups with DHA/EPA of 1.53–2.93 was significantly higher compared to group D/E0.55 while the activity of serum alternative complement pathway in group D/E2.93 was significantly lower compared with group D/E1.53. However, no difference was observed in activities of both respiratory burst of head kidney macrophage and serum catalase among dietary treatments. The per cent survival after air exposure in group D/E1.53, D/E2.08 and D/E2.93 was significantly higher compared with group D/E0.55. The fatty acid composition of whole body and tissues reflected closely those of diets, while fish accumulated more DHA than EPA in fish tissues, especially in muscle. These results suggested that at the same dietary contents of n‐3 LC‐PUFA (appr. 18% of TFA) and ARA (appr. 0.7% of TFA), moderate dietary DHA/EPA ratios of 1.53–2.08 significantly enhanced growth performances, certain innate immune responses, and the stress tolerance of Japanese seabass, in accordance with the preferential incorporation of DHA into fish tissues.     

Effects of diatom concentration in prepared feeds on growth and energy budget of the sea cucumber Apostichopus japonicus (Selenka)

Eight test diets were used in the experiment: seven fresh diatom (Cylindrotheca fusiformis) diets in which diatom occupied 100%, 83.3%, 50%, 33.3%, 20%, 14.3% and 11.1% in dry matter of the feeds (marked as D100, D83, D50, D33, D20, D14 and D11), and a formulated diet (70% Sargassum thunbergii powder, 20% sea mud and 10% fish meal, based on dry weight, marked as ST). The results showed that the specific growth rates (SGRs) of the animals fed diets D33, D20, D14 and D11 were not significantly different from the SGR of those fed diet ST. However, the energy allocated to growth for the animals fed with diet D14 was twice that of diet ST. For sea cucumbers fed diet D14 the largest expenditure part was allocated to respiration (55.4%), but for those fed diet ST, the largest part was allocated to faeces (62.7%). The organic content of diet D14 was also much lower than that of diet ST. Frozen diatom diets containing 14% (dry matter weight) fresh diatom could be an environmentally friendly feed as an alternative option for macroalgae powder.     

Effect of temperature on growth,digestion and immunity of green,white and purple sea cucumbers,Apostichopus japonicus (Selenka)

Three color morphs (white, green and purple strains) of Apostichopus japonicus (Selenka) were cultured in artificial seawater, for approximately 90 days, in three temperature ranges: 27–22°C (high), 22–17°C (mid) and 17–12°C (low). All strains grew in all temperature ranges. Temperature significantly affected growth rate, digestibility, digestive enzymes and immune‐related enzymes. Highest specific growth rates were exhibited in 4‐month‐old sea cucumbers at mid and high temperatures, and in 16‐month‐old sea cucumbers at mid and low temperatures. Specific growth rates of green and purple strains were not significantly different, but were significantly higher than that of the white strain at mid temperatures. The digestibility of each strain was significantly higher at 27°C, 22°C and 17°C than at 12°C. Green‐strain digestibility was higher than that of purple and white strains at specific temperatures. Protease and amylase activities of all strains followed bell‐shaped temperature curves with maximum digestive enzyme activity at 17°C. The activities of alkaline and acid phosphatases were higher in the guts of the green strain than in the white or purple strains at the same temperature. Superoxide dismutase activity was higher in the purple strain than in white and green strains.     

Effects of dietary n‐3 PUFA supplements on composition of n‐3 PUFA and expression of fatty acid elongase 5 (AJELOVL5) in sea cucumber,Apostichopus japonicus

This is the first comprehensive study on the effect of dietary polyunsaturated fatty acid (PUFA) levels on the expression of fatty acid elongase 5 (AJELOVL5), PUFA composition, and growth in juvenile sea cucumbers. The specific growth rate (SGR_w) was improved in n‐3 PUFA‐rich diets compared to low n‐3 PUFA diets. AJELOVL5 expression was apparently upregulated in juveniles fed lower PUFA diets relative to higher PUFA diets, with higher expression in the body wall and respiratory tree of juveniles fed diets without ɑ‐linolenic acid (ALA, 18:3n‐3) compared to juveniles fed higher ALA level diets; similar results were also detected in juveniles fed diets with lower eicosapentaenoic acid (EPA, 20:5n‐3), docosahexaenoic acid (DHA, 22:6n‐3), and none of ALA, EPA, or DHA respectively. The concentrations of ALA, EPA, and DHA in tissues were positively related to the content of dietary corresponding PUFA, with higher ALA content in juveniles fed diet ALA_12.71 than in the ALA_7.46 and ALA₀ groups. Similar results were also obtained in sea cucumber fed diets enriched with either EPA or DHA. Interestingly, considerable levels of EPA and DHA were found in the tissues of juveniles fed diets of CK₀ and DHA₀, with no specific input of EPA or DHA, showing that the sea cucumber was capable of biosynthesizing EPA and DHA from their corresponding precursors as ALA and linoleic acid (LA, 18:2n‐6).     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

Spontaneous captive breeding and larval development in the green and red variants of the Japanese sea cucumber Apostichopus japonicus (Selenka 1867)

We report the spontaneous spawning, larval development, survival rate and larval growth rate patterns observed in the green and red variants of the Japanese sea cucumber Apostichopus japonicus. The green variant adapted well to the captive conditions in the Sasebo City Fisheries Center and spontaneously spawned without any induction or stimulation. One hundred individual green variants spawned nine times and produced approximately 155 million eggs. In contrast, 50 individual red variants showed poor adaptation to captivity and spawned spontaneously only three times, producing about 12 million eggs. Larval development and growth rate pattern was almost identical between the two variants of A. japonicus. In contrast, the larval survival rate for the green variant was over 90% up to the auricularia stage (10 days), but much low (less than 30%) for the red variant. We demonstrated that the green variant of A. japonicus was easier to rear in captivity. This provides a useful method for maintaining sustainable harvests and eventually contributing to restocking and sea ranching of the existing population. Further studies about optimal ecological conditions and behaviour are needed to improve egg production and survival rate of the red variant of A. japonicus.