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.     

Effect of dietary chitosan oligosaccharide complex with Ce (IV) on growth,immunity and disease resistance against Vibrio splendidus of sea cucumber,Apostichopus japonicas

An 8‐week feeding trial was conducted to investigate the effects of dietary chitosan oligosaccharide complex with cerium (Ce IV) (COS‐Ce) on growth performance, nonspecific immunity and disease resistance of sea cucumber, Apostichopus japonicas. Five isonitrogenous (18.6%) and isolipidic (1.1%) practical diets were formulated with graded level of COS‐Ce (0, 150, 300, 600 and 1200 mg kg^?1 dry feed), which were named as COS‐Ce/0, COS‐Ce/150, COS‐Ce/300, COS‐Ce/600, COS‐Ce/1200 respectively. Each diet was allocated to four replicates of sea cucumbers (Initial weight: 6.72 ± 0.02 g). Sea cucumbers were fed to apparent satiation once daily (19:00 hours) for 56 days. During the experiment, water temperature was kept at 16 ± 0.5°C, pH 7.8–8.2, dissolved oxygen beyond 5 mg L^?1, ammonia nitrogen below 0.5 mg L^?1 and salinity from 30‰ to 31‰. Results showed that the specific growth rate of sea cucumbers was significantly higher in COS‐Ce/600 than that in other four treatments. Activities of phagocytosis, respiratory burst, acid phosphatase and alkaline phosphatase in COS‐Ce/600 were significantly higher than that in COS‐Ce/0 (P < 0.05) respectively. On the contrary, cumulative mortality was the lowest in COS‐Ce/600 following 14 days exposure to Vibrio splendidus (P < 0.05). In conclusion, these results confirmed that dietary COS‐Ce had beneficial effects on growth performance, nonspecific immunity and disease resistance of sea cucumber.     

Seasonal variations of immune parameters in the coelomic fluid of sea cucumber Apostichopus japonicus cultured in pond

In order to understand the effects of seasonal change on the immunity of sea cucumber Apostichopus japonicus cultured in pond, A. japonicus with body weight of 12.2 ± 4.5 g (sample A) and 32.6 ± 7.1 g (sample B), respectively, were collected monthly and randomly from a typical pond during a year cycle and employed for the evaluation of immunocompetence. Simultaneously, the environmental factors in the pond including water temperature, pH, salinity and dissolved oxygen (DO) were measured using a handheld multiparameter meter. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), lysozyme (LYZ), phenoloxidase (PO), superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) in the coelomic fluid were selected for the evaluation of A. japonicus immunocompetence and determined using biochemical methods. The results showed that in both samples, the activities of all determined enzymes had small values in winter and early spring, and LYZ, CAT and MPO activities also presented small values in summer, suggesting that pond‐cultured A. japonicus underwent immunosuppression twice during a year cycle, and the immunosuppression occurred in winter and early spring was more severe than that occurred in summer. In addition, most of the determined enzymes showed different variations between the two samples, and had significantly negative correlation with protein concentration, which was significantly and negatively correlated with water temperature, revealing that body weight and water temperature might have crucial effects on the immunity of A. japonicus cultured in pond.     

Requirement of vitamin E of growing sea cucumber Apostichopus japonicus Selenka

This feeding trial was conducted to determine the vitamin E requirement of growing sea cucumber Apostichopus japonicus Selenka. Six isonitrogenous and isoenergetic experimental diets were formulated to contain graded levels of vitamin E (6.7, 81.2, 159.3, 237.8, 314.6, 395.9 mg/kg diet). Each diet was assigned randomly to 30 growing sea cucumber with initial body weight 15.43 g in triplicates for 8 weeks. Survival rate was not affected by dietary vitamin E; meanwhile, both the weight gain (WG) and specific growth rate were presented the trend of increasing first and then kept stable. With the increasing of dietary vitamin E, crude lipid content of body wall was evaluated firstly and dropped afterwards. When dietary vitamin E contents were lower than 159.3 mg/kg, vitamin E contents of body wall were increased by dietary vitamin E levels, but there were no more differences when dietary vitamin E higher than 159.3 mg/kg. There were minor effects on digestive enzymes of intestine by dietary vitamin E. Contents of malondialdehyde were decreased, while the total superoxide dismutase activity was increased first and then decreased with the increasing of dietary vitamin E. Activities of intestinal alkaline phosphatase were increased, and glutamic oxaloacetic transaminase were decreased first and then increased with the increasing of dietary vitamin E. In conclusion, analysis by a linear regression equation of WG or vitamin E contents in body wall indicated that the optimum requirement of vitamin E for growing sea cucumber (initial body weight 15.43 g) was 165.2–187.2 mg/kg diet.     

免疫增强剂党参对仿刺参肠道菌群结构的影响

采用平板计数法和聚合酶链式反应( PCR)-变性梯度凝胶电泳( DGGE)技术分析免疫增强剂党参对仿刺参( Apostichopus japonicas)肠道菌群结构的影响。将初始体重为(18.00±2.00) g的仿刺参随机分为2组(对照组、试验组),每组6个重复,每个重复12只。对照组投喂海泥、鼠尾藻粉按照1∶1的质量比配制的饵料,试验组饵料中以鼠尾藻粉质量的2%添加党参,连续投喂28 d。结果表明：应用免疫增强剂党参不仅能够显著提高仿刺参的特定生长率( P<0.05),降低其饵料系数(P<0.05),而且能够显著增加仿刺参肠道内容物中异养菌的数量(P<0.05);序列统计分析显示投喂党参后仿刺参肠道细菌优质序列比例显著增加( P<0.05),试验组达97.57%,对照组仅为80.22%;Beta多样性分析反映投喂党参后仿刺参肠道微生态环境发生了变化,其多样性系数范围在14.91%~15.47%、15.47%~16.21%、14.91%~16.21%;丰度分析显示投喂党参后仿刺参肠道内容物中变形菌门( Proteobacteria)和拟杆菌门( Bacteroidetes)丰度提高,疣微菌门( Verrucomicrobia)、放线菌门( Actinobacteria)和厚壁菌门( Firmiaites)丰度降低;聚类分析显示试验组与对照组肠道菌群结构相似性系数为0.97。由此可见,免疫增强剂党参可提高仿刺参的特定生长率,降低饵料系数,增加肠道异养菌数量和优势菌群丰度,优化肠道微生态环境。     

刺参(Apostichopus japonicus)重要经济性状相关SNP标记的验证分析

本研究在前期构建的刺参(Apostichopus japonicus)高密度遗传连锁图谱和QTL分析的基础上，筛选出26个与体长、体重、体宽、棘刺总数、抗病力相关的SNP位点，设计出可用于HRM检测的SNP扩增引物13对。在扩大群体中利用HRM小片段法对这13个刺参重要经济性状相关的候选SNP位点进行分型和多态性检测，并结合扩大群体的相关性状数据进行了QTL位点验证。多态性结果显示，13个位点中有3个单态性位点，其余10个多态性位点中有3个位点为低等位多态性，7个位点为中等位多态性。10个多态性位点的最小等位基因频率(MAF)介于0.016(SNP113)~ 0.332(SNP160)之间，平均值为0.173；各位点的观测杂合度(Ho)介于0.031(SNP113)~0.818(SNP9)之间，平均值为0.433；期望杂合度(He)介于0.031(SNP113)~0.834(SNP9)之间，平均值为0.402；多态信息含量(PIC)介于0.030(SNP113)~0.393(SNP160)之间，平均为0.248，有6个位点偏离Hardy- Weinberg平衡。QTL验证结果表明，SNP40和SNP160位点为与生长(体长、体重、体宽)相关的位点，各位点的优势基因型分别为SNP40(CC)和SNP160(AA)；SNP88、SNP112和SNP126这3个位点为与抗病力相关的位点，各位点的优势基因型为SNP88(CC)、SNP112(AA)和SNP126(TT)。基于这5个位点构建生长和抗病二倍型，发现二倍型K1(CC AA TT)抗病力最强，S1(CC AA)、S3(CC AC)在生长方面优势显著，相关研究结果可为分子标记辅助育种在生产中应用提供基础数据。     

Nitrogen and phosphorus budget of a Haliotis discus hannai and Apostichopus japonicus polyculture system

The system nitrogen (N), phosphorous (P) budget, N/P utilization rate and the physiological response mechanism of the abalone Haliotis discus hannai (body weight: 12.87 ± 0.82 g) and the sea cucumber Apostichopus japonicus (body weight: 10.85 ± 1.16 g) to different co‐culture environment conditions were examined. Animals were kept in a multilayer, cubic recirculating aquaculture system at different polyculture densities (abalones at 400 ind/m²in monoculture [Group C] and abalones at 400 ind/m²with sea cucumbers at 10 ind/m² [AS1] or 20 ind/m² [AS2]). Each treatment was replicated four times, and the experimental cycle was 90 days. No significant difference in survival rate of abalones was detected when the stocking density of sea cucumbers increased from 10 to 20 ind/m², but the concentrations of total ammonia nitrogen and nitrite nitrogen in the water were significantly higher in AS2 than in AS1. Survival rate, specific growth rate (SGR) of body weight of sea cucumbers and SGR of body weight of abalones were significantly lower in AS2 than in AS1. No significant difference in protease (PES), lipase, amylase and cellulase activities of abalones was identified between Group C and AS1, but the PES and amylase activities of abalones and sea cucumbers in AS1 were significantly higher than those in AS2. In AS1, the N/P output from harvesting of abalones and sea cucumbers and the N/P utilization rates were significantly higher than those in AS2. Although the N/P output from faeces was significantly lower in AS2 than in Group C and AS1, the N/P output from the water layer was significantly higher than that in AS1. The expression levels of Cu/Zn superoxide dismutase (Cu/Zn‐SOD) and heat shock protein 70 (HSP70) of both abalones and sea cucumbers in AS2 were significantly higher than those in AS1. No significant difference in expression of catalase (CAT) and HSP90of abalones was identified among these groups, but the expression levels of CAT and HSP90of sea cucumbers in AS2 were significantly higher than those in AS1. These results indicate that stocking sea cucumbers at 10 ind/m² in the polyculture system will relieve the organic load on the system and improve the N/P utilization rate. It will also increase aquaculture production and improve the ecological and economic benefits of the system.     

3种水质调控方式下刺参池塘初级生产力的周年变化

通过对自然纳潮、微孔曝气、养水机池塘不同水层初级生产力及其相关参数的研究,分析养水机对初级生产力的影响。结果表明,3种水质调控方式池塘,初级生产力年均值、P/R值均以养水机池塘最高,微孔曝气池塘次之,自然纳潮池塘最低。养水机池塘、微孔曝气池塘、自然纳潮池塘的初级生产力年均值分别为(6.22±0.54)、(5.37±0.60)、(4.69±0.53) gO_2/(m~2·d)。3种水质调控方式下,养水机池塘30～50 cm水层和50～100cm水层初级生产力差异不显著,而微孔曝气池塘和自然纳潮池塘这两水层之间初级生产力差异显著,且养水机池塘50～100 cm的水层初级生产力显著高于微孔曝气和自然纳潮池塘。研究表明,养水机能显著提高刺参池塘50 cm以下水层的初级生产力,缩小上层和下层初级生产力之间的差距,从而提高池塘水体总初级生产力,为刺参饵料和池塘物质快速循环提供基本保障。