PHYSICOCHEMICAL CHARACTERISTICS AND ANTIOXIDANT EFFICACY OF CHITOSAN FROM THE INTERNAL SHELL OF SPINELESS CUTTLEFISH Sepiella inermis

Cuttlefish chitosan was extracted from the cuttlebone of Sepiella inermis by demineralization and deproteinization and produced by deacetylation, and its physical and chemical parameters were also compared with that of commercial chitosan. Ash, moisture, and mineral and metal content of the chitosan was estimated by adopting standard methodologies. The rate of deacetylation was calculated as 79.64% by potentiometric titration. Through viscometry and gel permeation chromatography, the molecular weight of chitosan was found to be significantly lower than that of the commercial chitosan. Optical activity was found to be levorotatory. The structure of the chitosan was elucidated with spectral techniques such as Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Cuttlefish chitosan showed a melting endothermic peak at 117.32°C. The x-ray diffraction (XRD) pattern of chitosan and standard chitosan exhibited the same crystalline peaks. Through scanning electron microscopy (SEM) the fine structure of chitosan was studied. The binding capacity (water and fat) of cuttlefish chitosan was found to be significantly higher than that of the commercial chitosan. The antioxidant efficacy of chitosan was determined through the conjugated diene method, scavenging ability on DPPH radicals, reducing power, and chelating ability on ferrous ions. This study has brought out the importance of shell as a potential source for obtaining another natural antioxidant.     

Ontogenetic trajectories of septal spacing in modern cuttlefishes are phylogenetically dependent

In this study, the ontogenetic trajectories of septal spacing between succeeding chambers of five modern cuttlefishes, Sepia esculenta, S. lycidas, S. latimanus, S. pharaonic and Sepiella japonica, which were all wild-caught around Japan, were analysed. The ontogenetic trajectories of septal spacing of all examined cuttlefishes demonstrate a decrease in septal spacing followed by an increase during the earliest ontogenetic stage. This trend is assumed to be related to hatching. After the rapid decrease and increase in septal spacing, species-dependent trends occur irrespective of sex and locality. Based on cluster analyses of general trends recognized in each species, the five examined species can be categorized into two groups: (1) a group by S. pharaonis, S. esculenta and Sepiella japonica; and (2) S. latimanus and S. lycidas as more distant branching groups within the five examined species. This classification is concordant with some phylogenetic clades determined from DNA analyses. Therefore, we hypothesized that the ontogenetic trajectories of septal spacing among modern cuttlefishes are phylogenetically dependent. If this hypothesis holds in fossil cuttlefishes, the examination of ontogenetic trajectories of septal spacing would give new insight into the recognition not only of the life history but also of the phylogeny of fossil cuttlefishes.     

饥饿胁迫对曼氏无针乌贼幼体的影响

以曼氏无针乌贼(Sepiella maindroni)初孵幼体为材料,分析了饥饿胁迫对幼体存活、行为变化,消化酶活力,脂肪酸组成,以及饥饿后再投喂对幼体生长及成活率的影响.结果表明:饥饿对曼氏无针乌贼幼体存活、体态和游动行为的影响较大,至第7天全部死亡.初孵幼体的胃蛋白酶活力在饥饿5d时出现最低值0.23±0.04 U·mg-1·prot-l;胰蛋白酶活力在饥饿5d时最高为737.30±26.26 U·mg-1·prot-1;淀粉酶活力在饥饿5d时降至最低13.93±2.00 U·g-1·prot-1;脂肪酶活力在饥饿2d时最低17.46±0.79 U·g-1·prot-1.饥饿状态下,机体消耗利用自身脂肪酸成分的顺序依次为:饱和脂肪酸,单不饱和脂肪酸,多不饱和脂肪酸中的低不饱和脂肪酸,最后是高不饱和脂肪酸.曼氏无针乌贼的不可逆点(PNR)为第5天.实验结果有助于合理制定曼氏无针乌贼育苗前期饵料的投喂策略.     

曼氏无针乌贼GT微卫星位点的筛选

采用链霉亲和素包被的磁珠富集法筛选曼氏无针乌贼(Sepiella maindroni)微卫星位点。试验样品来自舟山六横岛,提取4个样品的DNA混合成DNA pool,用限制性内切酶Sau 3A I酶切。接上接头后构建基因组PCR文库,用生物素标记的(GT)15探针筛选。将筛选获得目的片段进行PCR扩增,连接pMD18-T载体,转入DH5α感受态大肠杆菌里,扩大培养后PCR筛选阳性克隆。总共选取278个克隆,对120个经过检测含有插入片段的克隆进行测序,发现102个克隆含有微卫星序列,阳性克隆比率为85%。除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有64条。     

曼氏无针乌贼墨囊组织学及墨汁形成的超微结构

采用组织学和电镜技术对曼氏无针乌贼的墨囊及墨腺细胞进行了研究。结果表明:墨囊壁和导管壁由外膜、肌肉层和黏膜三部分组成;墨腺体集中在墨囊底部,呈索状,腺体中部含丰富的结缔组织;墨汁颗粒以游离态形式分布于索状腺体的间隙及墨囊腔中。实验观察到无分泌黑色素功能的A型细胞和有分泌黑色素功能的B型细胞;在B型细胞中可见黑色素颗粒储存在囊泡中,囊泡在移出细胞的过程中逐渐变大,泡内的黑色素颗粒逐渐变多。囊泡可能通过胞吐的方式排出细胞外,黑色素排出后以颗粒的形式游离于细胞间隙中,形成墨汁。     

三种乌贼内骨骼生化成分研究

曼氏无针乌贼（Sepiella maindroni）和金乌贼（Sepia esculenta）的内骨骼称为海螵蛸,可用于制酸、止血等。通过对曼氏无针乌贼、金乌贼和虎斑乌贼（Sepia pharaonis）内骨骼生化成分的比较,有望实现传统中药海螵蛸原料质量的提升和替代。分别使用烘干干燥法、高温灼烧法、凯氏定氮法、索氏提取法、酸碱浸泡法、茚三酮柱后衍生离子交换色谱法、离子发光色谱法检测水分、灰分、粗蛋白、粗脂肪、粗甲壳素、氨基酸及矿物质。结果显示：三种内骨骼营养成分中,虎斑乌贼水分和粗蛋白含量最高,分别为（2.52±0.16）%和（3.43±0.10）%;金乌贼灰分和粗脂肪含量最高,分别为（94.7±0.26）%和（0.24±0.04）%;甲壳素含量最高的为曼氏无针乌贼内骨骼,为（9.48±0.36）%,约为其他两种的1.50倍。三种乌贼内骨骼共检出15种氨基酸,其中必需氨基酸（EAA）7种,半必需氨基酸（HEAA）1种,非必需氨基酸（NEAA）7种,三种乌贼骨的EAA/TAA为44.64%~47.09%,符合FAO/WHO中对于蛋白质EAA/TAA的理想评定标准。金乌贼内骨骼的钙含量最高（3.93×10⁵ mg/kg）、曼氏无针乌贼的钠含量最高（1.02×10⁴ mg/kg）、虎斑乌贼的钾含量最高（589.5 mg/kg）。可见三种乌贼内骨骼均是低脂肪、高甲壳素、高矿物质的中药。虎斑乌贼内骨骼蛋白和灰分营养成分含量较为丰富,是否可以作为传统海螵蛸的替代品,值得进一步探讨。     

几种生态因子对曼氏无针乌贼野生和养殖卵孵化的影响

比较了曼氏无针乌贼野生卵和养殖卵的区别,研究了不同温度、盐度、孵化密度、卵类型对野生和养殖曼氏无针乌贼孵化率和孵化时间的影响.结果表明：野生卵的质量较佳,养殖卵则以黑色小卵的质量为佳.野生卵的最佳孵化温度为27 ℃～29 ℃,最佳孵化盐度为24.5～32.0;孵化密度对野生卵孵化率的影响不显著.养殖卵在19 ℃～29 ℃下的孵化率为6.7%～30.0%,高于33 ℃和低于17 ℃均不能孵化;在盐度19.5～32.0范围内孵化率为18.3%～25.0%,盐度低于17.0不能孵化;充气情况下,孵化密度对养殖卵的孵化率影响不显著,而不充气情况下影响显著.     

盐度胁迫下曼氏无针乌贼的行为和生理响应

盐度变化会引起头足类动物渗透调节、免疫能力和激素分泌异常等一系列生理应激反应，同时还会引发应激诱导的喷墨行为等异常行为变化，最终可能导致头足类因渗透失衡出现大量死亡。结合头足类苗种繁育过程中因海水盐度波动出现的摄食及行为异常等问题，探究了盐度胁迫对曼氏无针乌贼（Sepiella japonica）行为活跃性、组织结构、渗透调节以及免疫相关酶活性的影响，明确了其对盐度变化的应激反应及适应能力。结果显示：在高盐胁迫下曼氏无针乌贼的行为活跃性显著增加（P<0.05），且倾向于绕水槽壁游动；与21‰组相比，27‰组和33‰组肌肉、鳃和肝脏组织结构呈现较严重的损伤，15‰组的各组织结构无明显变化；随着盐度的升高，鳃中Na⁺/K⁺-ATP酶活性呈现先降低后升高趋势（P<0.05）；33‰组脑中皮质醇含量显著高于其他处理组（P<0.05）；盐度升高或降低均会导致鳃和肝脏中超氧化物歧化酶（Superoxide dismutase，SOD）、过氧化氢酶（Catalase，CAT）活性显著升高（P<0.05），丙二醛（Malondialdehyde，MDA）含量显著增加（P<0.05），溶菌酶（Lysozyme，LZM）活性显著降低（P<0.05）。综合分析认为，曼氏无针乌贼具有较好的耐低盐能力，高盐胁迫会引发其类焦虑行为。此外，在研究设定的15‰-33‰盐度范围内，盐度变化还会引起其渗透调节以及免疫相关酶活性的改变。研究结果为阐释头足类适应盐度波动的调节机理，控制适宜的养殖环境提供了参考依据。