温度对拟穴青蟹循环水养殖系统微生物群落结构的影响

为探究温度对拟穴青蟹(Scylla paramamosain)循环养殖水体中微生物群落的影响,深入理解环境因子与养殖水体微生物群落之间的相关性。在水温分别为18℃、22℃、26℃、30℃和32℃的循环养殖系统中进行拟穴青蟹养殖,采用16S rRNA高通量测序技术对不同温度下的养殖水体微生物群落组成进行分析。结果表明:(1) Alpha多样性指数显示5组水样中细菌群落多样性指数大小为t26t22t30t32t18,物种丰度大小为t22t26t32t30t18。温度可以导致养殖水体中微生物群落多样性发生变化,但并未发现二者之间直接的相关性。(2) 5种温度下的养殖系统中细菌群落共有31门、43纲、94目、185科和351属,变形菌门(Proteobacteria)为循环养殖水体中绝对优势菌门。在养殖系统水温为26℃时,养殖水体中氮磷污染物含量相对较高,其水中脱氮除磷菌属的相对丰度也显著高于其他4组。(3)由花瓣图和UPGMA聚类分析图进行相似性分析可知,水体微生物群落对温度的变化有着良好的响应, 5组水样的细菌群落结构总体相似性较低,其中最高温度32℃和最低温度18℃的水体中细菌群落结构最为相似。(4)水体中微生物群落结构和与环境因子的相关性分析显示,氮磷营养盐中的总磷(TP)和硝酸盐(NO3–-N)对水中微生物群落特征具有显著性影响,而温度是导致养殖水体中氮磷营养盐差异化的主要原因。上述研究结果初步解释了温度对拟穴青蟹循环养殖水微生物群落的作用机制,为下一步深入研究拟穴青蟹循环水养殖系统中环境变化与微生物的相互关系提供参考。     

Mud crab pen culture: replacement of fish feed requirement and impacts on mangrove community structure

Brackishwater pond culture has been a major factor in mangrove loss in Southeast Asia, hence, the need to develop environment‐friendly technologies such as mud crab Scylla (Portunidae) culture in mangrove pens exists. This study evaluated the effects of mud crab netpen systems in central Philippines on mangrove macroflora, and the replacement of dietary fish with low‐cost pellets. Wild or hatchery‐sourced Scylla olivacea and Scylla serrata were stocked at 0.5–0.8 m⁻² in 167–200 m² nylon netpens (2.3 cm stretched mesh) in Avicennia‐dominated mangrove habitats. The feeding treatments were: (A) Zarraga: (1) no feeding (natural productivity), (2) no feeding for 1 month+supplementary feeding, (3) fish biomass and (4) low‐cost pellets, and (B) Batan: (1) fish biomass and (2) pellets+fish biomass. Feeds were given ad libitum twice daily. Growth and survival rates of S. olivacea in Zarraga pens were not significantly different among treatments, although crabs fed fish biomass had the highest survival, body weight and production. Similarly, growth and survival of S. serrata were not significantly different between the Batan treatments. Economic analysis of the latter gave a 38.5% return on investment (ROI) and 2.6 years payback period (PP) for pellets+fish biomass treatment compared with 27.5% ROI and 3.6 years PP for fish alone. Sensitivity analysis showed an improved economic performance of the pellets+fish biomass treatment by increasing the survival rate. Evaluation of mangrove community structure showed that crab culture reduced species diversity, numbers and biomass of seedlings and saplings, but not of mangrove trees. Therefore, mud crab pen culture is recommended for mangrove sites with mature trees, but not seedlings and saplings, and low‐cost pellets can reduce dependence on fish biomass.     

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20–33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.     

拟穴青蟹不同发育时期胚胎基因表达的内参基因的筛选

为获得适用于研究拟穴青蟹(Scylla paramamosain)不同发育时期胚胎基因表达的内参基因,本研究采用3个内参基因筛选软件geNorm、NormFinder及BestKeeper对微管蛋白α1A(Tuba1a)、核糖体蛋白L13(Rpl13)、泛素(UBQ)、核糖体蛋白S6激酶(Rps6)、精氨酸激酶(Ak)、3-磷酸甘油脱氢酶(gapdh)、28S核糖体RNA(28S)、18S核糖体RNA(18S)和延伸因子1A基因(EF-1α)等9个常用候选内参基因在拟穴青蟹不同发育时期胚胎的表达稳定性进行分析。geNorm分析表明, EF-1α和Rpl13的表达稳定性最高,并且采用2个内参基因同时校正目标基因的定量结果可以达到最优效果; NormFinder分析表明, gapdh和EF-1α是9个候选内参基因中表达最稳定的; BestKeeper分析表明,gapdh的标准偏差和变异系数是9个候选基因中最低的。综合3个内参基因筛选软件,可以选用EF-1α,或选用EF-1α和gapdh双内参同时校正拟穴青蟹不同胚胎发育时期各基因的表达情况。     

Quality assessment of newly hatched mud crab,Scylla serrata,larvae

Starvation and exposure to formalin were investigated as possible stress tests for evaluating the quality of mud crab, Scylla serrata, larvae. For the starvation stress test, newly hatched zoeae stocked in 150‐ml containers were either starved or fed rotifers. Similarly, newly hatched zoeae were stocked in containers with seawater of 0 (control), 20, 30 and 40 mg/L formalin for the formalin stress test. The zoeae from the same batches were used for seed production to monitor their performance and validate the results of stress tests. Starvation was found to be unsuitable for larval quality evaluation. However, the impact of initial food deprivation on the newly hatched larvae indicates that feeding immediately after hatching is necessary for mud crab larvae. Exposure of larvae to 40 mg/L formalin for 3 hr appeared to be a reliable and practical method for larval quality assessment as the survival of larvae in the mass production tanks validated the classification of good and poor quality batches in the stress tests. On this basis, a hatchery operator can decide which batch should be cultured further. Finally, there appears to be a link between the quality of larvae and the performance at the megalopa and early juvenile crabs.     

林下珍稀药用植物蛇足石杉繁殖的研究进展

蛇足石杉系石杉科石杉属多年生草本植物,内含石杉碱甲,为乙酰胆碱脂酶的有效抑制剂,是治疗阿尔茨海默病症的特效药。随着世界人口老龄化的加速,蛇足石杉的需求量不断增加,然而蛇足石杉是古老的蕨类植物,其野生资源极其匮乏,而其繁殖却极其困难,为给蛇足石杉快速繁殖体系的研究与建立提供参考依据,文中综述了有关蛇足石杉的组织培养、孢子诱导、扦插繁殖、芽胞繁殖等方面的研究进展,并对其人工快速繁殖研究的新方向进行了展望。     

Effects of diets supplemented with either individual or combined probiotics,Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, on the immune response and disease resistance of the mud crab,Scylla paramamosain (Estampador)

Two trials, including firstly, diets incorporating individual or combined species of probiotics, Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, were fed to the mud crab, Scylla paramamosian, for 28 days to evaluate the growth, immunity and disease resistance, and secondly, the optimal level of B. subtilis E20 in the diet by tracking the growth, immunity and disease resistance after 28 days of feeding were analysed. No significant differences in growth, total haemocyte counts, respiratory bursts, superoxide dismutase, or glutathione peroxidase were found in the two trials. Crabs fed the B. subtilis E20‐containing diet (D20) had significantly increased phenoloxidase (PO) activity, phagocytic activity (PA) and disease resistance compared with crabs fed the control and the mixed probiotics diet (MD). The mortality of crabs fed the D20 was significantly lower than that of crabs fed the L. plantarum 7‐40‐containing diet. L. plantarum 7‐40 had a great inhibitory effect on the growth of B. subtilis E20, which may have led to the decreased probiotic effect of the MD. An analysis of the optimal level of B. subtilis E20 in the diet showed that crabs fed the B. subtilis E20‐containing diet at a level of 10^9–10 cfu kg^?1 had better immune response of PO and PA, and disease resistance in the second trial.     

室外虾池笼养拟穴青蟹（Scylla paramamosain）的存活、蜕壳与生长

本文介绍了在对虾养殖池中笼养青蟹的研究结果。养殖试验历时60 d,以存活率、蜕壳率、甲长增长率、甲宽增长率和增肉系数为指标参数,比较了不同材质、容积和放置层次的养殖容器对笼养青蟹存活、蜕壳与生长的影响。结果表明：在水温25～30℃、盐度13～20、 pH7.8～8.3条件下,笼养青蟹的存活率为（59.85±9.50）%、蜕壳率为（40.69±5.90）%、甲长增长率为107.73%、甲宽增长率为89.39%、增肉系数为286.11%、肥满度＞1。养殖容器的材质、容积和放置层次对笼养效果有影响：胶绳网箱明显优于（ P〈0.05）塑料箱;容积10 L组与6 L组、18 L组相比养殖效果最佳,差异显著（ P〈0.05）, S组略优于B组,差异不明显（ P＞0.05）;第二层（中层）笼箱与第三层（底层）、第一层（表层）相比,笼养效果最佳,差异极为显著（P〈0.01）,第三层（底层）笼箱略优于第一层（表层）,但差异不明显（P＞0.05）。研究结果显示了在对虾养殖池内实施笼养青蟹方式的可行性。     

锯缘青蟹血蓝蛋白抑菌活性及其作用靶标

采用抑菌实验和抑菌抑制实验等方法探索锯缘青蟹(Scylla serrata)血蓝蛋白抑菌活性及其作用靶标.结果显示,锯缘青蟹血蓝蛋白对溶藻酸弧菌(Vibrio alginolyticus)、大肠杆菌K12(Escherichia coli K12)等细菌具有抑菌活性,其对大肠杆菌K12的抑菌活性可被大肠杆菌K12外膜蛋白(Outer membrane protein, Omp)所抑制,尤其是血蓝蛋白对大肠杆菌K12外膜蛋白敲除菌ΔOmpX、ΔOmpC、ΔOmpT、ΔTsx、ΔFadL和ΔOmpW的抑菌活性显著降低,与对照组相比,具有极显著性(P<0.01)和显著性差异(P<0.05).由此说明,锯缘青蟹血蓝蛋白具有抑菌活性,其抑菌作用靶标可能为多种外膜蛋白.