Phylogeny of the cuttlefishes (Mollusca: Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA geneare sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae , is not supported. From the two families ofSepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA andamino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, butdo not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNAof cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at ahigher taxonomic level (i.e., family).

Isolation via enrichment and characterization of ten polymorphic microsatellite loci in the cuttlefish, Sepiella maindroni de Rochebruns

Ten novel polymorphic microsatellite loci were developed using an enrichment and colony hybridization protocol and characterized for the cuttlefish,Sepiella maindroni.Polymorphism was explored by using 30 individuals from the coastal waters of Zhanjiang,Guangdong province,China,in December 2007.The number of alleles ranged from 5 to 13.The ranges of observed and expected heterozygosity were from 0.578 9 to 1.000 0 and 0.682 8 to 0.925 7,respectively,and the average polymorphic information content （P IC） was 0.778 5.These microsatellite loci will certainly facilitate the detection of the genetic variation and population structure of S.maindroni.

Isozymes Analysis of the Golden Cuttlefish Sepia esculenta (Cephalopoda: Sepiidae)

Thirty nine isozymes in four tissues (mantle muscle, buccal bulb muscle, eye and liver) of Sepia esculenta were screened for enzymatic analysis using starch gel electrophoretic technique. Eighteen enzymes (G3PDH, LDH, MDH, MEP, IDHP, PGDH, GRS, NP, AAT, CK, AK, EST, ALP, ACP, FBP, MPI, GPI and PGM) show strong activities and good convergence in zymogram. They are proved to be suitable genetic markers in Sepia esculenta. Among the tissues used, mantle muscle is the best for electrophoretic analysis of isozymes. Eye and liver are fairly good for some special enzymes, such as LDH, EST, MPI, etc. Twenty six loci are detected. The proportion of polymorphic loci is 0.115 in the Qingdao sample and 0.153 in the Rizhao sample ( P < 0.99 ). The mean values of the observed and expected heterozygosity per locus of Qingdao sample are 0.016 and 0.017 , while those of the Rizhao sample are 0.023 and 0.025 respectively.

BIOCHEMICAL GENETIC STUDIES ON CUTTLEFISH SEPIELLA MAINDRONI (CEPHALOPODA: SEPIIDAE)──ACTIVE LOCI SCREENING OF ISOZYME

Screening of 46 putative enzyme coding loci and 4 different kinds of tissues of Sepiella maindroni de Rochebrone, 1884 for enzymatic activities using starch gel electrophoretic technique proved that the 21 enzymes such as AAT, AK, ALP, AP, CK, DIA, ES, FBP, G3PDH, GPI, GRS, IDH, LDH, MDH, MEP, MPI, NP, PGDH, PGM, SOD and XO *, were active to Sepiella maindroni after being stained. The tissue exhibiting stable and clear bands was also determined. Among tissues tested, mantle muscle tissue was the best for electrophoretic survey of isozymes. Buccal bulb muscle, eye and liver were fairly good for some special enzymes, such as DIA, ES, MPI, NP, etc.

Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Liver of Golden Cuttlefish <i>Sepia esculenta</i>

The distribution of chitinolytic enzymes in eight organs of the golden cuttlefish Sepia esculenta was determined. Chitinase activity (activity of endo-type chitinolytic enzyme) was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates, with high activity detected in the liver, posterior salivary gland, and stomach. β-N-acetylhexosaminidase (Hex) activity (activity of exo-type chitinolytic enzyme) was determined using pNP-(GlcNAc) as a substrate, and high activity was observed in six organs, including the liver, branchial heart, posterior salivary gland, and stomach. In addition, two chitin-binding proteins (CBP-A, CBP-B) were isolated from the liver using a chitin affinity column. Two full-length cDNAs (SeChi-1: 1484 bp;SeChi-2: 1748 bp) encoding chitinases were obtained from the liver of S. esculenta. SeChi-1 contained a 1377-bp open reading frame (ORF) encoding 459 amino acids, and SeChi-2 contained a 1656-bp ORF encoding 552 amino acids. Domain structures predicted from the deduced amino acid sequences of SeChi-1 and SeChi-2 (SeChi-1, SeChi-2) contained signal peptides, a GH Family 18 catalytic domain, one chitin binding domain (CBD) in SeChi-1, and two CBDs in SeChi-2. Proteome analysis revealed that 125 peptide residues of CBP-A were present in SeChi-1, and 116 peptide residues of CBP-B were present in SeChi-2. Organ expression analysis revealed that SeChi-1 and SeChi-2 were expressed only in the liver of S. esculenta. Phylogenetic analysis of SeChi-1, SeChi-2, and GH family 18 chitinases revealed that SeChi-2 belongs to a group of previously reported squid chitinases, while SeChi-1 does not belong to any previously reported group of mollusk chitinases.

Changes in embryonic development,juvenile growth and physiological adaptation of the cuttlefish Sepia pharaonis in response to photoperiod manipulation

Photoperiod is a key environmental indicator for re gulating embryonic development,individual growth and physiological processes in aquatic animals.In this study,differences in embryonic development and performance of newly hatched cuttlefish juvenile exposed to five different cycles of light:dark(L:D):constant light,18 L:6D,12 L:12D,6 L:18D cycles,and constant darkness were evaluated.Prolonged exposure to light induced an accelerated rate of embryonic development,particularly after the red-bead stage.Principal component analysis(PCA)revealed that red-bead stage,heartbeat,endoskeleton formation,pigment appear,and six increments of cuttlebone were the main factors contributing to the embryonic development.Meaning that the duration time of these five stages were significantly different when exposed to photoperiod regimes,which may determine the duration of the incubation period of the embryos.Long term light has also affected the incubation parameters with an increased rate in hatching and shortened the incubation and hatching periods in the 12-24-h day length range.However,constant light and darkness environment appeared to have a greater effect on the stress of embryonic development,mainly reflected in the yolk shed ratio and the inking rate in the egg capsule.Moreover,the increase in the day length has contributed to improve the growth and survival of juveniles in the 12-18-h day length range;however,juveniles exposed to constant light and darkness experienced worse re sults in terms of growth,tissue glycogen content,dige stive enzymes of the digestive glands,and metabolic enzymes of the muscles.These finding suggest that prolonged light exposure accelerates the process of embryonic development,maximum feeding time is not necessarily a condition of optimal growth,and inappropriate light cycles can disturb the body’s endogenous controls.Therefore,the optimal photoperiod for the embryos development and juvenile growth of Sepia pharaonis were 12 h and 12-18 h of day length,respectively.These results are useful for increasing the production of this species during embryo incubation and juveniles rearing in aquaculture practice.

Effects of Different Weaning Protocols on Survival,Growth and Nutritional Composition of Pharaoh Cuttlefish(Sepia pharaonis)Juvenile

The aim of this study was to determine the optimal weaning protocols for rearing juvenile cuttlefish Sepia pharaonis,as well as to understand the feeding habits of cuttlefish and develop effective aqua-cultural practices.The effects of four experimental weaning protocols(i.e.,food type,cuttlefish size,food ration(bw d^−1)and feeding frequency(meals d^−1))on the growth,survival and muscle proximate composition of cuttlefish juvenile were studied in quintuplicate.The weaning period lasted 2 weeks.The results showed that cuttlefish fed with frozen white shrimp(Exopalaemon carinicauda)displayed a significantly higher survival,growth parameters,ingestion rate,food conversion rate,and muscle protein content when compared with the other prepared frozen foods.The larger individuals revealed better growth performance than smaller individuals,especially in terms of survival and specific growth rate,indicating that larger individuals were more receptive to frozen prey.Furthermore,size grading should be conducted to ensure better growth during weaning.As food rations increased,the survival,growth and muscle protein content of juveniles were promoted,whereas the most appropriate food ration for cuttlefish juvenile was 35%bw d^−1.Similarly,higher feeding frequency could stimulate the appetite of animals reared on weaning,and enhance survival,growth,and protein deposition compared to low feeding frequency.These results suggest that the optimal feeding protocols of juvenile weaning is as follows:the ideal prepared food is frozen shrimp,the weight of juveniles should be 4.0 g(32 days old)or more,the food ration should be 35%(bw d^−1),and the optimum feeding frequency is 4 meals day^-1.This study offers a promising starting point to successfully breed this candidate aquaculture species.

Identification and Expression Profile of a Neuropeptide LFRFamide-Like Gene During Different Stages of Gonadal Development in the Cephalopod Sepia pharaonis

Neuropeptides are widely distributed in vertebrates and invertebrates,regulating a variety of physiological activities in the organisms,such as metabolism,feeding and reproduction.In this study,to explore the function of neuropeptide LFRFamide in Sepia pharaonis,the full-length cDNA of LFRFamide-like gene(named SpLFRFL,MG869822.1)was identified with rapid amplification of cDNA ends(RACE)method.The sequence of SpLFRFL was 860 bp in length and encoded 188 amino acids containing 4 different mature peptides:1 copy of PHTPFRFamide,NSLFRFamide,TIFRFamide,and 3 copies of GNLFRFamide.Multiple alignment and phylogenetic analysis results showed that SpLFRFL shared high identity with LFRFamides of Sepia officinalis and Sepiella japonica and had the closest relationship with them.Through quantitative Real-time PCR(qRT-PCR),it was found that the SpLFRFL gene was highly expressed in the optic lobe and brain at three different stages during gonad development in both genders.Moreover,the four mature peptides at a concentration of 0.01μmol L^(−1) could inhibit the protein synthesis in the Chinese hamster ovary cell strain-K1(CHOK1)induced by SpGnRH.These data suggest that SpLFRFL might be involved in the development and reproduction of S.pharaonis.The results can contribute to future studies on neuropeptide evolution and function and benefit the cuttlefish farming.

墨鱼骨结构的力学行为及其应变率效应

墨鱼骨是一种墨鱼内部产生的生物矿化壳,通过调节壳内的气液比从而实现墨鱼的深浅浮动,同时满足轻质和高刚度的力学特性,使墨鱼能够很好地适应深海环境,所以墨鱼骨是一种典型的高比刚度的多孔材料。为探究墨鱼骨结构的力学性能,通过Instron材料试验机和分离式Hopkinson压杆实验装置,对墨鱼骨在不同加载应变率下的力学行为进行研究。研究结果表明,墨鱼骨在准静态加载下,其应力-应变曲线呈现典型的三阶段变化模式,即弹性段、平台段和压实段,具有很好地吸能缓冲作用;随着加载应变率的提高,墨鱼骨的初始压溃应力和平台应力也随之增加,表明墨鱼骨材料对加载应变率存在很强的敏感性。进一步分析不同生长方向的墨鱼骨在准静态压缩下的力学行为,结果表明随着生长方向的增加,墨鱼骨结构的刚度和吸能性能都得到了弱化,从而揭示了墨鱼骨材料压缩行为的各项异性。

Determination of fatty acids percentages and profile extracted from cuttlefish of Iranian coasts of Persian Gulf and Oman Sea

Objective:To determine the fatty acid profile extracted from cuttlefish of Persian Gulf and Oman Sea,including Ancistrocheirus,Enoploteuthidae,Cranchiidae,Ommastrephidae and Loliginidae.Methods:Oil was extracted by the Bligh and Dyer method.The fatty acid profile of the oil was determined by gas chromatography.Results:The results showed that(13±5)%of wet weight of cuttlefish is oil.The results also showed that cuttlefish oil has 29.40%saturated fatty acids and 23.70%single-band unsaturated fatty acids,and the total value of the unsaturated multiple-band is 40.20%,the contents of arachidonic acid 2.78%,linolenic acid 3.10%,linoleic acid 5.20%,docosahexaenoic acid 15.40%,and eicosapentaenoic acid 9.60%out of the total fatty acids of the same may be mentioned.Conclusions:The results of the current study described that cuttlefish is considered for the first time as the new and rich source of omega-3 and omega-6.

Cuttlefish Egg Soup

The cuttlefish egg is in fact the eggs-binding gland of cuttlefish. It is elliptical and grows on the internal surface of ovary, with a layer of thin film coating the gland. Inside the gland are many small white "cuttlefish coins" which nestle closely to each other, To cook cuttlefish egg soup, prepare the cuttlefish coins by peeling each one carefully so they keep their original shape, then

2种乌贼肌肉的营养成分及品质评价

为了探究2种乌贼肌肉营养成分及品质差异,评价其营养价值,本研究对浙江舟山地区捕获量较大的日本无针乌贼(Sepiella japonica)和金乌贼(Sepia esculenta)的常规营养成分、质构特性、蒸煮损失率、甲醛含量、氨基酸和脂肪酸组成及矿物元素进行了分析。结果显示,日本无针乌贼肌肉的粗蛋白、粗脂肪含量与金乌贼相比无显著差异(P>0.05),水分含量显著低于金乌贼(P<0.05),粗灰分含量显著高于金乌贼(P<0.05)。金乌贼肌肉的硬度、弹性、胶粘性、咀嚼性和内聚性均显著高于日本无针乌贼(P<0.05)。2种乌贼肌肉的甲醛含量分别为0.56和1.18mg/kg,均符合国家卫生标准。氨基酸测定结果显示,2种乌贼的肌肉中均检测出17种水解氨基酸,第一限制性氨基酸均为色氨酸;其中,必需氨基酸占总氨基酸的比例均高于31%,金乌贼与FAO/WHO推荐的理想蛋白模式更接近,其必需氨基酸指数(EAAI)高达82.99。脂肪酸测定结果显示,2种乌贼的肌肉中均检测出20种脂肪酸;其中,C20:5n-3(EPA)和C22:6n-3(DHA)的总含量均高于40%,且日本无针乌贼显著高于金乌贼(P<0.05)。2种乌贼肌肉富含人体所需的多种无机元素,常量元素中K和P含量最高,微量元素中Zn和I含量最高。研究表明,2种乌贼肌肉的营养成分及品质存在一定的差异,但均属于优质的低脂蛋白,具有较好的开发利用价值。本研究为开发利用东海头足类海洋资源提供了一定的依据和参考。

基于围压装置的墨鱼骨试验和分析

为了研究墨鱼骨在深海环境中的适应性和力学行为,设计了围压装置,并通过围压装置来模拟深海压力,结合材料试验机进行试验。试验结果表明,在稳定的围压下,墨鱼骨的应力应变曲线呈现出三个阶段,围压状态下屈服应力明显大于大气压下,由此验证墨鱼在深海环境中能够更好地承受压力。通过研究墨鱼骨在围压状态下的力学行为,为设计和制造高性能深海设备材料提供新思路。

静电纺丝制备非彩虹结构色膜

本研究提出了全新的制备非彩虹结构色膜方法,并且利用静电纺丝工艺,首次在静电纺丝溶液体系中添加诸如石墨烯、墨鱼黑等黑色物质,制备出非彩虹结构色膜。研究证明黑色物质的添加对于在静电纺丝溶液体系中制备非彩虹结构色膜具有普适性。此外,对制得的结构色膜的微观形态,反射光谱,变角反射光谱进行了检测。

乌贼游动机理及其在仿生水下机器人上的应用

针对大多数机器鱼未采用弹性机制来提高能量利用效率和耐压能力低等的不足,对拥有高超游动能力,具有耐压结构的乌贼进行研究。分析乌贼喷射和鳍波动推进的游动机理,给出喷射推力、喷射和整周期流体推进效率方程。乌贼复合游动方式的优点是高速性和低速性均很好,能瞬时改变游动方向,噪声低,以及即使喷射速度低于周围流体速度,也能产生推力。为深入说明乌贼游动机理,研究乌贼外套膜和鳍这两套运动系统的肌肉性静水骨骼结构及其动作原理。肌肉性骨骼不但具有支撑躯体,进行动作和输出力的作用,还具有良好的耐压能力。大多数乌贼体内没有充气组织,外套膜腔内外静压平衡,进一步提高了它们的耐压能力。乌贼动作时,弹性机制能够减少能量消耗,提高能量利用效率。若能将乌贼的游动方式、肌肉组织结构和弹性机制等特点应用到仿生水下机器人上,将使其更加高效、灵活和耐压。

不同干燥方法对乌贼品质及微观结构的影响

为了解不同干燥方法对乌贼干后品质及微观结构的影响,采用自然晾晒干燥、热风干燥和真空冷冻干燥3种方法对乌贼进行处理,研究其对乌贼感官特征、色泽、收缩率、风味、甲醛含量和微观结构的影响,以及在后续复水过程中复水率和复水完成后质构参数的变化。结果表明,干制后乌贼的收缩率变化为:自然晾晒干燥>热风干燥>真空冷冻干燥,且差异显著(P<0.05)。真空冷冻干燥的乌贼在色泽、复水率方面优于其他两组,在第9小时的纯水复水率可达524.25%,为自然晾晒干燥组的2.5倍,而在质地和风味方面的品质一般,其微观肌肉纤维形变较小,纤维间孔径较大;热风干燥后的样品在感官、质地以及风味方面具有显著优势,呈味氨基酸含量总和达1 519.20 mg/100 g,显著高于其他干燥组(P<0.05),但样品中的甲醛含量较高,为260.83 mg/kg,是新鲜样品的4.7倍,且肌肉纤维形变大,排列致密;自然晾晒干燥的乌贼肌肉纤维形变较小,排列较致密,其甲醛含量较低,仅为22.06 mg/kg,感官品质和色泽还有待进一步提高。

乌贼墨对小鼠免疫功能的影响

本文报道乌贼墨对小鼠免疫功能影响的初步研究结果。实验组和对照组小鼠腹腔巨噬细胞对鸡红细胞吞噬率(55.8±9.5％和32.1±5.1％)和吞噬指数(3.12±0.41和2.38±0.50)均有明显差异。用ELISA法检测两组小鼠对肿瘤细胞抗原刺激所产生的血清特异性抗体量分别为4.43±0.78(log2)和3.17±0.86(log2),(P<0.01)。并且前者的脾脏重量大于后者,胸洙重量则无明显差别。

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。

乌贼墨多糖提取工艺优化

通过单因素实验和正交实验,对乌贼墨多糖的提取工艺进行了研究。主要考察了不同料液比、pH、提取温度以及提取时间对多糖得率及含量的影响,同时比较了活性炭吸附和高速离心法对乌贼墨黑色素的去除效果。实验结果表明:高速离心法去除黑色素方法优于活性炭吸附;乌贼墨多糖最佳提取工艺条件为:料液比为1∶2,pH为5,提取温度为40℃,提取时间为4h,此条件下乌贼墨多糖得率为1.228%,糖含量为78%,初步纯化后蛋白脱除率为90.52%,多糖的损失率为12.45%。

金乌贼胚胎发育的研究

对人工孵化条件下金乌贼的胚胎发育进行了观察。刚产出的卵子,均重0.99g/粒(n=100),长轴为11～24mm,短轴为8～15mm,卵黄如鸡蛋状,梨形,长径5～6mm,短径3～4mm,外包三级卵膜。三级卵膜在胚胎发育过程中经历一个柔软-定形-缩小-膨大的变化过程。受精卵在20～25℃水温下孵化,经过26d幼体陆续脱膜孵出。根据胚胎发育的外部形态及典型特征将金乌贼胚胎发育过程划分为细胞分裂期、囊胚期、原肠期、器官发育期、出膜期5个典型时期。在胚胎发育过程中,眼的形态变化比较大,且经历一个无色、红色再到黑色的变化过程。