Rheological and Structural Properties of Sea Cucumber Stichopus japonicus During Heat Treatment

Changes in tissue structure, rheological properties and water content of raw and heated sea cucumber meat were studied. Sea cucumber Stichopus japonicus was heated at 25℃, 70℃ and 100℃ water for 5 min. The structural changes were observed using a light microscope and the rheological parameters （rupture strength, adhesive strength and deformation） determined using a texture meter. Microscopic photograph revealed that the structural change of heated meat was greater than that of raw meat. The rupture strength, adhesive strength and deformation of raw meat were smaller than those of the heated meat. Meanwhile, rheological parameters showed positive correlation with heating temperature. These changes are mainly caused by thermal denaturation and gelatinization of collagen during heating. These changes were also evidenced in observations using a light microscope and differential scanning calorimetry.     

Antioxidation Activities of Low-Molecular-Weight Gelatin Hydrolysate Isolated from the Sea Cucumber Stichopus japonicus

Gelatin extracted from the body wall of the sea cucumber (Stichopus japonicus) was hydrolyzed with flavourzyme.Low-molecular-weight gelatin hydrolysate (LMW-GH) of 700-1700 Da was produced using an ultrafiltration membrane bioreactor system.Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner;IC50 value for superoxide and hydroxyl radicals was 442 and 285 μg mL-1,respectively.LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells.Furthermore,LMW-GH notably increased intracellular glutathione (GSH),which in turn suppressed melanogenesis.LMW-GH performs antioxidation activity,holding the potential of being used as a valuable ingredient in function foods,cosmetics and pharmaceuticals or nutriceuticals.     

Antiviral effects of <Emphasis Type="Italic">Stichopus japonicus</Emphasis> acid mucopolysaccharide on hepatitis B virus transgenic mice

Hepatitis B virus（HBV） is a significant global pathogen and efficient cure for HBV patients is still a challenging goal. We previously reported that acidic mucopolysaccharide from stichopus japonicus selenka（SJAMP） could inhibit HBs Ag and HBe Ag expression in vitro. However, the potential anti-HBV effects of SJAMP in vivo have not yet been explored. In this study, we show that SJAMP exhibits potent anti-HBV activity in HBV transgenic mice in a dose-dependent manner. Specifically, sixty HBV transgenic male BALB/c mice were randomly selected to receive the treatment of PBS, low dose SJAMP(30 mg kg^-1), middle dose SJAMP(40 mg kg^-1), high dose SJAMP(50 mg kg^-1) and IFN(45 IU kg^-1) for 30 d. SJAMP treatment suppressed serum HBV-DNA, and liver HBs Ag and HBc Ag levels in HBV-transgenic mice. The present study highlights the potential application of SJAMP in HBV therapy.     

Fat-1转基因模型小鼠对脂多糖诱导的抑郁样行为和中枢炎症反应的改善作用

以Fat-1转基因小鼠和野生型C57BL/6小鼠为研究对象,经侧脑室注射脂多糖(lipopolysaccharide,LPS)诱导中枢炎症模型,测定抑郁样行为糖水偏好度和悬尾实验、腹腔巨噬细胞活性及其吞噬能力、脑炎症小胶质细胞的标记物CD11b基因与蛋白的表达水平,用以研究Fat-1对LPS诱发的炎症和抑郁样行为的作用。结果表明:在野生型小鼠中,LPS降低小鼠糖水偏好度,延长悬尾静止不动时间,增加腹腔巨噬细胞的活性和吞噬能力,上调CD11b基因和蛋白表达水平;而LPS不能引起Fat-1转基因小鼠出现上述类似变化。侧脑室注射LPS可引起野生型小鼠炎症反应和抑郁样行为,而Fat-1转基因小鼠可通过脑内和体内合成n-3 PUFAs抵抗LPS诱发的上述抑郁样变化。     

Effects of Different Microbes on Fermenting Feed for Sea Cucumber (Apostichopus japonicus)

The effects of different microbes on fermenting feed for sea cucumber(Apostichopus japonicus) were compared to select the optimal fermentation strain in this study. Saccharomgces cerevisae, Candida utilis, Bacillus subtilis and Geotrichum candidum were independently added into the experimental compound feed, while only saline was mixed with the control feed. The fermentation treatments were inoculated with 10% seed solution under the condition of 25℃ and 70% water content, which lasted for 5 days to elucidate the optimal microbe strain for fermenting effect. Physicochemical indexes and sensorial characteristics were measured per day during the fermentation. The indexes included dry matter recovery(DMR), crude protein(CP), the percentage of amino acid nitrogen to total nitrogen(AA-N/t N), the percentage of ammonia nitrogen to total nitrogen(NH3-N/t N), and the ratio of fermentation strains and vibrios to the total microbes, color, smell and viscosity. The results showed that DMR, CP and AA-N/t N of the S. cerevisae group reached the highest level on day 3, but the ratio of fermentation strain was second to C. utilis group. In addition, its NH3-N/t N and the ratio of vibrios were maintained at low levels, and the sensory evaluation score including smell, color and viscosity was the highest in S. cerevisae group on day 3. Therefore, S. cerevisae could be the optimal strain for the feed fermentation for sea cucumber. This research developed a new production method of fermentation feed for sea cucumber.     

Effects of photoperiod on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka)

In nature, Apostichopus japonicus exhibits a behavioral characteristic of emerging at night and sheltering during the day. Hence, it was commonly believed that longer and darker light conditions are better for a population of A. japonicus. In aquaculture situations therefore, animals have commonly been cultured without lighting, especially during juvenile-hatching culture. However, how the length of darkness each day affects A. japonicus and what the mechanism is of behavioral response of A. japonicus to different photoperiods remain unclear, and are specifically addressed in this study. We applied nine photoperiod treatments (light:dark (LD) cycles at 0:24, 3:21, 6:18, 9:15, 12:12, 15:9, 18:6, 21:3, and 24:0 under 500 lx) to 60 individuals (29.73 ± 0.23 g) per treatment (with 3 replicates). The environmental conditions were set as: water temperature = 16.9 ± 0.3°C, dissolved oxygen>6.0 mg/L, ammonia<0.3 mg/L, pH = 7.8-8.2, and salinity = 30-32. Under experimental conditions of continuous darkness as well as continuous light, cyclic nocturnal activity patterns of A. japonicus (viz. the animals emerged and fed at night and sheltered during the day) were observed. However, they spent more and less time, respectively, moving and feeding under continuous darkness and continuous light, than those under a natural light cycle. Under photoperiods with 6-12 h of light, the animals showed one sheltering behavior transition and two emerging behavior transitions. These behavior transitions appeared to be governed by some internal physiological factors and induced by daily light variation. The behavior of the animals was significantly affected by different photoperiods, and the distribution rate (DR) of the animals emerging at "daytime" increased with lengthening "light time" (except for the LD 3:21 cycle). We also found that there was no significant difference in mean DR per day under LD cycles of 6:18, 9:15, 15:9, and 12:12. Mean DRs per day were significantly lower for these photoperiods, than for those under LD cycles of 0:24 and 3:21, and significantly higher than for those under LD cycles of 18:6, 21:3 and 24:0. These results imply that 6-15 h light per day were the similar photoperiods to natural sunlight. From the viewpoint of activity and feeding behaviors, these might be the optimal photoperiods for rearing A. japonicus.     

Effects of environment factors on initiation of sperm motility in sea cucumber Apostichopus japonicus (Selenka)

Sperm of sea cucumber Apostichopus japonicus (Selenka) were quiescent in electrolyte NaCl solution and artificial seawater (ASW) and nonelectrolyte glucose and mannitol solutions when the osmolality was less than 200 mOsm kg-1. The sperm started to be motile as a result of increased osmolality, indicating an osmolality-dependent initiation of sperm motility in sea cucumber. After a brief incubation in hypotonic NaCl and glucose solutions with osmolalities of 200 and 400 mOsm kg-1, sperm lost partial motile ability. Sperm became immobilized when pH was 6.0 in NaCl, glucose and mannitol solutions, suggesting that an H+ release is involved in sperm activation. The decreased pH had no effect on the percentage of motile sperm in ASW, whereas it delayed the time period to reach the maximum motility (motilitymax). Extracellular Ca2+ in electrolyte solutions was not essential for motility stimulation but shortened the time of reaching motilitymax. When Ca2+ was mixed in nonelectrolyte solutions the sperm motility was completely suppressed. The K+ channel blocker, quinine, suppressed the sperm motility in electrolyte solution, showing a possible involvement of K+ transport in the process. High K+ concentration did not affect the sperm motility in NaCl solution, but decreased it in ASW and almost entirely suppressed it in nonelectrolyte solutions. The different effects of pH and K+ in ASW and NaCl solution indicate that external ions may also regulate sperm motility.     

溶藻弧菌肽聚糖对凡纳滨对虾白斑综合征病毒的抑制作用

从患病凡纳滨对虾(Litopenaeus vannamei)体内提取白斑综合征病毒(WSSV),PCR证实WSSV毒性,将病毒粗提液稀释10-2、10-3、10-4和10-5倍,攻毒实验表明,最佳攻毒剂量为10-4倍稀释液。对凡纳滨对虾(Litopenaeus vannamei)分别注射0.05、0.10、0.15 mg/m L的溶藻弧菌(Vibrio alginolyticus)肽聚糖,免疫24 h后感染WSSV,测定其免疫保护率和肝胰腺的免疫学指标,探讨溶藻弧菌肽聚糖对凡纳滨对虾抗WSSV的影响。结果表明:对虾的保护率随着肽聚糖的浓度增大而增大,与对照组相比,0.05、0.10、0.15 mg/m L组的保护率分别为36.67%、46.67%、56.67%;实验组凡纳滨对虾肝胰腺中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和一氧化氮合成酶(NOS)活力与对照组相比的差异有统计学意义(P0.01)。溶藻弧菌肽聚糖可有效增强凡纳滨对虾自身的非特殊性免疫能力,提高抗WSSV的能力。     

外源视黄酸对草鱼早期胚胎发育的影响

以 10 - 6 M和 10 - 5 M的视黄酸体外处理草鱼 ( Ctenopharyngodon idellus)早期胚胎 ,结果发现其影响了胚胎的形态发育 ,影响其中轴器官的形成和神经系统的分化 ,使胚胎前部结构全部或部分缺失 ,由此诱导的神经器官 ,如眼、听囊、嗅囊等缺失或不分化 ,胚胎的尾部缩短 ;同时也减少了色素细胞的形成。并且这种畸形程度随着处理浓度加大 ,时间加长而加剧。但在原肠中期以后处理的胚胎 ,神经系统发育基本正常 ,因此说明视黄酸并不抑制原肠作用和随后的神经诱导。它可能是通过抑制某些基因的表达从而影响神经外胚层的区域性分化。     

维生素E醋酸酯对鱼油脂肪酸含量变化的影响

新鲜鱼油中添加05、0、2505、00 mg/kg 4种不同浓度的维生素E醋酸酯,在实验的第6、14、28、36和68天测定鱼油中的脂肪酸含量。结果表明:在500 mg/kg添加量内,维生素E醋酸酯对鱼油中脂肪酸含量没有显著性影响(P>0.05)。在实验期间,EPA(C20:5)、DHA(C22:6)、亚油酸(C18:2)含量明显下降,ARA(C20:4)、亚麻酸(C18:3)先升后降,棕榈酸(C16:1)逐步上升;C14含量逐渐增加,C17则先降后升,C16和C18在实验后期有较大提高。维生素E醋酸酯对鱼油中不饱和脂肪酸效用系数大小依次为:ARA>EPA>DHA>C18:3>0>C16:1>C18:2>C18:1,对ARA抗氧化效用最大,效用系数达到10.193%,C20:5为0.490%,C22:6为0.364%;维生素E醋酸酯对饱和脂肪酸效用系数大小依次为:C14>C17>0>C18>C16。     

Effects of water depth and substrate color on the growth and body color of the red sea cucumber,Apostichopus japonicus

Three color variants of the sea cucumber,Apostichopus japonicus are recognized,the red one is highly valued in the market. When the red variant is cultured in ponds in China,its body color changes from red to celadon in 3–6 months. The effects of water depth and substrate color on the growth and body color of this animal were investigated. Juveniles of red A. japonicus were cultured in cages suspended at a range of water depths(20,50,100,150 and 200 cm). The specific growth rate of red sea cucumbers was significantly higher in animals cultured at deeper water layers compared with those grown at shallowers. Body weights were greatest for sea cucumbers cultured at a depth of 150 cm and their survival rates were highest at a depth of 200 cm. A scale to evaluate the color of red sea cucumbers(R value) was developed using a Pantone standard color card. All stocked animals in the 9-month trial retained a red color,however the red body color was much more intense in sea cucumbers cultured at shallower depths,while animals suspended in deeper layers became pale. In a separate trial,A. japonicus were cultured in suspended cages with seven different colored substrates. Substrate color had a significant effect on the growth and body-color of red A. japonicus. The yield were greatest for A. japonicus cultured on a yellow substrate,followed by green white orange red black and blue. All sea cucumbers in the 7-month trial retained a red color,although the red was most intense(highest R value) in animals cultured on a blue substrate and pale(lowest R value) for animals cultured on a green substrate.     

Localization and Dynamic Expression of a 27.8kDa Receptor Protein for Lymphocystis Disease Virus Infection in Sea Bass(Lateolabrax japonicus) Tissues

Lymphocystis disease virus(LCDV) infects target cells by attaching to a 27.8 k Da receptor(27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies(MAbs) have been developed. However, the 27.8R existence in tissues of sea bass(Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.     

Effects of spatio-temporal and environmental factors on distribution and abundance of wintering anchovy Engraulis japonicus in central and southern Yellow Sea

We investigated the spatio-temporal and environmental factors that affect the distribution and abundance of wintering anchovy and quantifi es the infl uences of these factors. Generalized additive models(GAMs) were developed to examine the variation in species distribution and abundance with a set of spatiotemporal and oceanographic factors, using data collected by bottom trawl surveys and remote sensing in the central and southern Yellow Sea during 2000–2011. The fi nal model accounted for 28.21% and 41.03% of the variance in anchovy distribution and abundance, respectively. The results of a two-step GAM showed that hour, longitude, latitude, temperature gradient(TGR), and chlorophyll a(Chl- a) concentration best explained the anchovy distribution(presence/absence) and that a model including year, longitude, latitude, depth, sea surface temperature(SST), and TGR best described anchovy abundance(given presence). Longitude and latitude were the most important factors affecting both distribution and abundance, but the area of high abundance tended to be east and south of the area where anchovy were most likely to be present. Hour had a signifi cant effect on distribution, but year was more important for anchovy abundance, indicating that the anchovy catch ratio varied across the day but abundance had an apparent interannual variation. With respect to environmental factors, TGR and Chl- a concentration had effects on distribution, while depth, SST, and TGR affected abundance. Changes in SST between two successive years or between any year and the 2000–2011 mean were not associated with changes in anchovy distribution or abundance. This fi nding indicated that short- and long-term water temperature changes during 2000–2011 were not of suffi cient magnitude to give rise to variation in wintering anchovy distribution or abundance in the study area. The results of this study have important implications for fi sheries management.     

Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber (Apostichopus japonicus)

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.     

Effects of rearing temperature and density on growth, survival and development of sea cucumber larvae, Apostichopus japonicus (Selenka)

In laboratory conditions, effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively. Data series like larval length and density, metamorphic time, and survival rate of the larvae were recorded. Statistics showed that for A. japonicus, survival rate (from fertilized egg to late auricularia) decreased significantly with the increasing rearing temperature (P<0.05). At different temperatures SGR was statistically significant as well (P<0.05) from day 1, and maximal SGR was found on day 9 at 24°C (159.26±3.28). This study clearly indicated that at low temperature (<24°C), metamorphic rate was remarkably higher than at higher temperature (>26°C). Hatching rate was significantly different between 0.2–5 ind./ml groups and 20–50 ind./ml groups. Rearing larvae at the higher density had the smaller maximal-length, whereas needed longer time to complete metamorphosis. This study suggested that 21°C and 0.4 ind./ml can be used as the most suitable rearing temperature and stocking density for large -scale artificial breeding of A. japonicus’s larvae.     

Effects of the Dietary Probiotic Clostridium butyricum on Intestine Digestive and Metabolic Capacities,SCFA Content and Body Composition in Marsupenaeus japonicus

A 56-day feeding trial was performed to evaluate the effects of dietary probiotic Clostridium butyricum(CB) on intestine digestive and metabolic capacities, intestine short-chain fatty acids(SCFA) content and body composition of kuruma shrimp Marsupenaeus japonicus. Shrimp s were randomly allocated into 9 tanks, 30 each, and f ed with diets containing different levels of C. butyricum(1×10~9 cfug~(-1)): 0 mgg~(-1) feed(Control), 100 mgg~(-1) feed(CB-100), 200 mgg~(-1) feed(CB-200), while each level was triplicated. The results indicated that compared with the control group, the intestine pepsin(Pep) activity and 5-hydroxytryptamine(5-HT) concentration of two C. butyricum group s were both increased. Amylase(AM Y) and lipase(LPS) activities wer e only induced in CB-200 group. Alanine aminotransferase(ALT) and aspartate aminotransferase(A ST) activities of two C. butyricum group s showed no significant change. The α-amylase(AM Y) gene expression was induced in CB-200 group, and tryp sin gene expression of two C. butyricum treated group s were both induced. Intestine SCFA content and body composition analysis showed that the contents of propionic acid, butyric acid and the crude protein of two C. butyricum group s were all higher than those of control. These results revealed that C. butyricum can modulate intestine digestive and metabolic capacities, improve intestine SCFA content and body crude protein content in M. japonicus.     

Effects of Nutritional Factors on the Growth and Heterotrophic Eicosapentaenoic Acid Production of Diatom Nitzschia laevis

The effects of several nutritional factors on the growth and eicosapentaenoic acid (EPA) production of diatom Nitzschia laevis were studied. 4 LDM (quadrupled concentration of the nutrient salt) was the optimal concentration of nutrient salt for the growth and EPA production ofN. laevis. The growth ofN. laevis was inhibited when the glucose concentration was either lower than 10gL-1 or higher than 15gL-1. Both sodium nitrate and urea were good nitrogen sources for the growth and EPA production, while ammonium chloride seriously decreased the dry cell weight (DW) and the EPA content. Silicate seriously influenced the growth of N. laevis. The maximum DW of 2.34gL-1 was obtained in the presence of 150 mgL-1 Na2SiO3·9H2O. The EPA content remained almost the same when the silicate concentration was lower than 150 mgL-1; however, higher silicate concentrations resulted in a steady de- crease of EPA content. Low medium salinity (≤29) did not seem to influence the DW of N. laevis, and high salinity resulted in a decrease of DW. The highest EPA content (4.08%) and yield (110 mgL-1) were observed at the salinity of 36 and 29, respectively.     

溶藻弧菌肽聚糖和vp28-siRNA对凡纳滨对虾白斑综合征病毒的抑制作用

对感染白斑综合征病毒(WSSV)的凡纳滨对虾(Litopenaeus vannamei)分别注射0.05和0.10μg/μL的vp28-si RNA,干扰效果实验表明,si RNA最佳浓度为0.10μg/μL。凡纳滨对虾感染WSSV 24 h时注射0.10μg/μL的vp28-si RNA,检测免疫后不同时间点的干扰效果。结果表明:在注射vp28-si RNA 6 h后,实验组与对照组WSSV病毒拷贝数差异有统计学意义(P0.05);在48 h时实验组病毒拷贝数急剧下降,对照组则保持较高水平,可较好干扰WSSV病毒复制,干扰效果持续120 h,对凡纳滨对虾的保护率为40%。凡纳滨对虾用0.10 mg/m L肽聚糖(Peptidoglycan)免疫24 h后感染WSSV,检测6、12、24 h注射vp28-si RNA的保护率。结果显示:6 h注射组的保护率为80%,12 h注射组为63.33%,24 h注射组为56.67%。凡纳滨对虾在肽聚糖和vp28-si RNA作用后,可增加其对WSSV抗感染作用,降低死亡率,干扰时间越早,对对虾的保护率越高。     

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber(Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0(control), 10~5, 10~7, and 10~9 CFU g~(-1) for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10~9 CFU g~(-1)(P 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber(P 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g~(-1) inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10~9 CFU g~(-1) not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.