曼氏无针乌贼胚胎发育的初步观察

将曼氏无针乌贼受精卵卵膜剥离,在显微镜或解剖镜下观察胚胎发育的全过程,结合室内人工育苗的孵化技术进行探讨。曼氏无针乌贼卵为粘性卵,呈黑色,类型属端黄卵,卵膜属三级卵膜,卵裂类型为不全卵裂,囊胚呈盘状,发生类型为直接发生型。在水温为23.8～26.2℃、盐度29.3时,最快14d孵出幼体,最慢23天孵出幼体,孵出幼体高峰期为18～21d,高峰期的孵出量占总孵出量的79.4%,孵化率为83.1%。胚胎发育速度与温度高低（20～30℃）成正比,盐度低于22对孵化率有严重影响。     

曼氏无针乌贼胚胎发育生物学零度和有效积温的研究

通过室内恒温培育试验和数理统计方法，研究了曼氏无针乌贼（Sepiella maindroni）受精卵发育的生物学零点温度、有效积温及胚胎发育的温度系数（Q10）。结果表明，曼氏无针乌贼受精卵发育的生物学零点温度为（6．48±0．44）℃；受精卵发育为初孵幼体的有效积温为（396．91±2．81）℃·d；18．0～24．0℃为曼氏无针乌贼胚胎发育的最适温度范围。     

光照对曼氏无针乌贼行为习性的影响

设置了3个光照强度梯度和红、白、橙、绿、蓝5个光照颜色,研究光照对曼氏无针乌贼行为习性的影响。试验结果表明:曼氏无针乌贼对光色和光强都有反应,而且表现出正趋性;对于同一光强下的红、白、橙、绿4种光照颜色来说,曼氏无针乌贼较喜欢红光和绿光。     

金乌贼和曼氏无针乌贼胚胎发育及其盐度耐受能力的比较研究

采集金乌贼(Sepia esculenta)及曼氏无针乌贼(Sepiella maindroni)受精卵,观察了金乌贼胚胎发育过程,比较分析了2种乌贼受精卵和仔乌的形态特征;分别研究了金乌贼受精卵在盐度20,25,30(对照)、33,36及曼氏无针乌贼受精卵在盐度15,20,25,30(对照)、36条件下的孵化率,分析了2种乌贼受精卵在192 h盐度突变过程中卵液渗透压和受精卵Na~+/K~+-ATPase活力的变化,比较了其在相同盐度20,25,30,36条件下的孵化率及耐受盐度突变的生理适应过程.结果表明,金乌贼胚胎发育第7天器官开始分化,第14天器官开始形成,盐度30、水温22～24℃时,其孵化时间为20～21 d;盐度30处理组,2种乌贼受精卵的孵化率均显著高于其他盐度处理组(P<0.01);在192 h盐度突变过程中,2种乌贼受精卵卵液渗透压随环境盐度的变化而变化,金乌贼受精卵卵液渗透压与环境渗透压相等,而曼氏无针乌贼受精卵卵液渗透压比环境渗透压平均高出60 mOsm/kg,2种乌贼受精卵均未检测到Na~+/K~+-ATPase活性.比较发现,虽然2种乌贼受精卵调节渗透压能力均较弱,但曼氏无针乌贼受精卵比金乌贼能更好地适应盐度变化.本研究旨在探明两种乌贼受精卵对盐度的适应性,为开展规模化人工繁育与资源增殖提供基础依据.     

曼氏无针乌贼对溶解氧耐受力的研究

在封闭的容器内,设置不同的温度梯度,进行不同规格的曼氏无针乌贼对溶解氧耐受力的实验.采用隔膜法检测实验过程中不同阶段的溶解氧,及结合乌贼养殖生产实际情况,研究其对溶解氧耐受情况.结果表明曼氏无针乌贼耗氧率与水温成正比例关系、与个体体重成反比例关系,耗氧率为146.0～431.4mg/kg·h-1,窒息点为1.96～3....     

胶州湾曼氏无针乌贼资源量与季节变化的调查研究

根据2005年和2006年逐月在胶州湾进行的底拖网调查，并对底拖网渔获物中的头足类进行定性定量分析与测定，利用扫海面积法评估胶州湾海域曼氏无针乌贼的资源量。调查分析了其分布特点与季节变化，并进行了渔获物中头足类组成的比较，分析了其种类组成、分布特点与季节变化特征。结果表明，胶州湾曼氏无针乌贼资源量有明显的季节变化，夏、秋两季的资源量明显高于其他季节，分别达到26.7t和21.5t（2006）；夏、秋两季的重量比明显高于其他季节，分别达到23.4%和27.8%。近几年，以曼氏无针乌贼为主捕对象的头足类资源量基本保持稳定，年平均资源量约14t，接近20世纪90年代中期的水平。     

曼氏无针乌贼的卵子发生及卵巢发育

采用组织学方法对人工养殖曼氏无针乌贼(Sepiella maindroni)卵子发生、卵巢发育周期进行组织学、细胞学观察。根据细胞大小、胞核形态及卵母细胞与滤泡细胞的关系,将曼氏无针乌贼卵子发生划分为卵原细胞期、卵母细胞期、成熟期和退化吸收期4个阶段,并阐述了各期卵母细胞的组织学特征。曼氏无针乌贼卵子发生过程具有种的特异性,大约孵化出膜12 d的乌贼可见有卵原细胞,此后进入增殖期。卵母细胞属于典型的滤泡型,发育过程中同时存在同步性与异同步性;卵母细胞无初级卵膜,只有次级卵膜和三级卵膜,次级卵膜由滤泡细胞分泌,三级卵膜由输卵管腺、缠卵腺以及墨囊分泌物共同构成。通过对卵巢的外观形态和组织学观察,将曼氏无针乌贼卵巢发育划分为6个时期。     

杂色鲍一种编码CRAD功能域基因的克隆及其在发育和应激中的表达

为探讨Caspase募集功能域(CRAD)在鲍发育和应激中的作用,实验通过比对杂色鲍转录组序列和RACE扩增的方法,获得了杂色鲍一条含CRAD的基因全长c DNA,命名为Hd CRAD;采用实时定量PCR的方法获得了该基因在发育和应激中的表达谱。结果显示,Hd CRAD的c DNA全长1 371 bp,开放阅读框735 bp,编码244个氨基酸。编码蛋白具有保守的CRAD功能域。该基因在各检测组织中均可表达,在黏液腺中表达量最高;在发育各阶段也均有表达,面盘幼虫晚期表达量最高。在细菌感染、高温或缺氧应激处理后,血细胞的Hd CRAD表达水平均有显著性变化。在担轮幼虫时期,RNA干扰该基因会显著提高幼虫畸形率;幼虫的caspase-8和DAD1的表达水平会显著上升,而caspase-3的表达水平会下降。研究表明,该基因参与了鲍幼虫发育过程;在成体免疫、耐高温和抗低氧中发挥了一定作用。     

野生和养殖曼氏无针乌贼亲体形态、生化组分及其胚胎发育的比较

为优化曼氏无针乌贼人工繁育技术,提高人工增殖苗种的产量与质量,采用实验生态学方法,对比分析了野生和养殖曼氏无针乌贼（Sepiella japonica）的表观形态、生化组分及其受精卵的形态差异,探究了光照周期与受精卵胶质外膜对曼氏无针乌贼胚胎发育的影响。结果显示：野生和养殖曼氏无针乌贼的表观形态、生化组分均存在显著差异,野生群体性成熟规格远大于养殖群体,肌肉和肝脏组织中的脂肪含量显著低于养殖群体（P<0.05）,卵巢中的蛋白质含量则显著高于养殖群体（P<0.05）;养殖亲体所产黑卵在12D12L光照周期条件下孵化率最高,所产白卵在24D和24L条件下孵化率最高,野生亲体所产卵在24D和12D12L条件下孵化率最高;随光照时间的延长,受精卵的平均孵化时间具有先增加后减小的趋势,表明光照周期是影响曼氏无针乌贼胚胎发育的重要因素;实验发现,将受精卵胶质外膜剥离处理后,养殖亲体所产黑白卵的平均孵化时间具有减少的趋势,但孵化率显著下降（P<0.05）。综上所述,建议在曼氏无针乌贼人工苗种生产过程中,尽量选用大规格野生乌贼作为繁育亲体。此外,在受精卵孵化期间,应对孵化池进行适当遮光处理,避免受精卵长时间暴露于强光照环境中,以提高孵化率和仔乌成活率。本实验研究结果可为曼氏无针乌贼人工苗种繁育技术优化提供参考。     

亚硝酸氮对曼氏无针乌贼幼体的急性毒性及免疫系统的影响

研究采用常规生物毒性实验方法,进行了亚硝酸氮对曼氏无针乌贼幼体的急性毒性实验,测定了在不同浓度亚硝酸氮急性毒性胁迫下,曼氏无针乌贼幼体内酸性磷酸酶(ACP),碱性磷酸酶(ALP)、超氧化物酶活性(SOD)和过氧化氢酶(CAT)的变化,并对其致死浓度进行了测定。结果表明,96 h的急性毒性实验对曼氏无针乌贼幼体的存活和免疫系统的活性有明显影响。在亚硝酸氮胁迫下,ACP活性在0.03 mg/L时急剧上升到最高,随胁迫浓度的增高,活性逐渐下降,在3.34 mg/L时低于对照组,在最高浓度胁迫下活性降到最低;ALP活性随亚硝酸氮胁迫浓度的增高也出现先增加后降低的趋势。SOD活性在亚硝酸氮胁迫下逐渐升高,在0.03 mg/L时达到最高,随后逐渐下降,但直到6.67 mg/L时才低于对照组;CAT活性也在0.03mg/L时即达到最高,但在0.67 mg/L时即低于对照组,并在最高浓度时降到最低。本实验还得出,亚硝酸氮的LC50(mg/L)为3.71 mg/L,安全浓度(mg/L)为0.013 mg/L。     

Growth differentiation factor 9 of Megalobrama amblycephala: molecular characterization and expression analysis during the development of early embryos and growing ovaries

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factorβ superfamily and plays an essential role during follicle maturation in mammals. In the present study, the full-length complementary DNA (cDNA) of gdf9 was obtained from Megalobrama amblycephala. The cDNA sequence is 2,061 bp in length with an open reading frame of 1,287 bp encoding 428 amino acid residues. The deduced amino acid sequence shared identities of about 42–86 % with the homologues of other vertebrates. During the early development of embryos, the gdf9 mRNA was detected in zygote with significantly high level and declined sharply by 47 and 87 % at 4 hours post-fertilization (hpf) and 6 hpf and even to an undetectable level through advancing stages. Expression analysis based on quantitative real-time PCR revealed that gdf9 mRNA was mainly expressed in ovary, but much lower levels were also found in some nonovarian tissues. Within the follicle, gdf9 mRNA was localized both in the oocytes and the follicle layer cells by in situ hybridization. During the ovarian cycle, gdf9 mRNA significantly decreased after the previtellogenic stage and became to increase again after the fully grown stage. The results imply that Gdf9 may play critical physiological functions in M. amblycephala early embryonic development and reproduction.     

饥饿对曼氏无针乌贼肝脏与卵巢脂肪酸组成的影响

在曼氏无针乌贼（Sepiella maindroni）性腺成熟阶段（4～5月）对其进行饥饿处理，以空腹处理组饥饿0d（禁食1d）、饥饿12d（禁食13d）和饥饿24d（禁食25d）为取样时间取其肝脏和卵巢组织进行脂肪酸组成及质量分数测定。结果表明，随着饥饿时问的延长，乌贼体质量下降，肝脏和卵巢萎缩，肝脏中的饱和脂肪酸（SFA）和单不饱和脂肪酸（MUFA）较空腹处理组有显著下降（P〈0．05），而多不饱和脂肪酸（PUFA）和高不饱和脂肪酸（HUFA）均有显著提高（P〈0．05）。与肝脏相比，乌贼卵巢中的各脂肪酸组成在饥饿12d变化较小，饥饿24d时∑SFA较空腹处理组有极显著下降（P〈0．01），而∑MUFA、∑PUFA＋∑HUFA和∑n-6 PU—FA显著高于空腹处理组（P〈0．05）。     

曼氏无针乌贼缠卵腺组织学及超微结构的研究

采用组织学和电镜技术对曼氏无针乌贼的缠卵腺及其腺体细胞进行了研究。试验结果表明,缠卵腺由缠卵腺壁、分泌叶瓣两部分构成。其中分泌叶瓣由两类细胞组成:Ⅰ型细胞和Ⅱ型细胞,Ⅰ型细胞有分泌功能,分泌物以糖蛋白为主;Ⅱ型细胞无分泌功能,对Ⅰ型细胞起支持作用。     

黄条鰤hsp70基因克隆及其在早期生长发育过程中的表达调控特性

热休克蛋白(heat shock proteins, HSPs)在鱼类的应激与免疫反应中发挥重要的生理调控作用,HSP70是该家族的重要成员。为探讨热休克蛋白在大洋性经济鱼类黄条鰤 (Seriola aureovittata)生长发育中的生理作用,本研究克隆获得了黄条鰤hsp70基因的全长cDNA序列,并采用定量PCR技术测定了其组织分布及在早期生长发育过程中的表达特征。结果显示,黄条鰤 hsp70基因的cDNA序列全长为2 332 bp,其中,5′-UTR长度为187 bp,ORF长度为1 920 bp,3′-UTR长度为225 bp,编码639个氨基酸,蛋白质分子量为70.1 kDa,等电点为5.16。黄条鰤 hsp70 mRNA的组织表达具有性别二态性差异,其中,在雌性鳃、心、脾脏和卵巢组织中显著高表达(P<0.05),且以卵巢中表达量最高;雄性垂体、鳃、头肾和精巢组织显著高表达(P<0.05),且以鳃中表达量最高。胚胎发育过程的表达检测显示,在卵裂前的受精卵中可检测到hsp70的表达,表明其具有亲本遗传的特性。同时,在胚胎发育过程的各个时期都可检测到hsp70 mRNA的表达,且在低囊胚期之前的各发育阶段一直保持较低表达水平,在原肠前期开始显著上调表达(P<0.05),其后保持相对较高表达水平,至胚胎孵化出膜期达峰值。在仔稚幼鱼中,hsp70 mRNA在初孵仔鱼和1 d仔鱼中高表达,其后在4 d仔鱼中显著降低(P<0.05),其后显著上调表达,至15 d仔鱼达峰值,其后在20 d仔鱼显著下降,并在25 d后稚鱼和幼鱼中保持相对较低表达水平。研究结果可为深入认识黄条鰤hsp70基因的结构特征、发生发育及其早期生长发育阶段的表达调控功能提供依据。     

Molecular characterization of cholecystokinin in grass carp (Ctenopharyngodon idellus): cloning, localization, developmental profile, and effect of fasting and refeeding on expression in the brain and intestine

Cholecystokinin (CCK) is a multi-functional brain–gut peptide in fish and mammals. To investigate the role of CCK in appetite regulation in fish, a 770-bp full-length cDNA sequence of CCK gene was obtained by RT-PCR and rapid amplification of cDNA ends methods in grass carp Ctenopharyngodon idellus. Homology analysis showed that the CCK cDNA sequence of grass carp had the highest similarity (90 %) to that of goldfish Carassius auratus and a higher similarity (>70 %) to those of other teleosts than to mammals. The PCR amplification using genomic DNA identified that the CCK gene of grass carp was comprised of three exons and two introns. Real-time quantitative PCR was used to detect CCK mRNA expression in adult tissues. High levels of gene expression were found in the hypothalamus and pituitary; moderate levels in the intestine, muscle and white adipose tissue; and low levels in other tissues. During early development (i.e., fertilized eggs to 35-day post-hatching larvae) the levels of CCK mRNA expression were higher during embryonic developmental stages than during post-hatch larval stages. Fasting decreased CCK mRNA expression levels in the brain and intestine, whereas refeeding resulted in an increase of expression. The results suggest that CCK mRNA expression has obvious tissue specificity and may have a role in feed intake regulation in grass carp.     

Molecular cloning and expression analysis of P-selectin glycoprotein ligand-1 from zebrafish (Danio rerio)

To date, the best characterized glycoprotein ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1). In this study, we cloned the full-length cDNA of PSGL-1 from zebrafish (Danio rerio). Zebrafish PSGl-1 cDNA is 1,594 bp and encodes a putative 284 amino acid protein with a theoretical molecular weight of 30.33 kDa and isoelectric point of 7.96. A signal peptide of 27 amino acids is predicted. The putative protein contains an extracellular mucin-like domain, a transmembrane domain and a cytoplasmic domain, with homology to mammalian PSGL-1. In the putative P-selectin binding region, there are 1 potential tyrosine sulfation site and 12 potential threonine O-glycosylation sites. A single extracellular cysteine, at the junction of the extracellular and transmembrane domains, suggests a disulfide-bonding pattern. The amino acid sequence of zebrafish PSGL-1 is 19–22% identical to that of mammalian PSGL-1. RT–PCR and whole-mount in situ hybridization analysis revealed that zebrafish PSGL-1 was expressed in early embryonic development, and the expression has an increased trend from 0.2 (1-cell stage) to 72 hpf. The results indicate that the general domain structure of PSGL-1 protein is conserved among species, and zebrafish PSGL-1 plays important roles in embryonic development and probably has similar biological function to that of mammalian PSGL-1.     

曼氏无针乌贼副性腺中6种酶的初步研究

采用试剂盒测定了曼氏无针乌贼副性腺产卵前后乳酸脱氢酶、碱性磷酸酶、酸性磷酸酶、过氧化氢酶、过氧化物酶和超氧化物歧化酶的活力,比较其产卵前后的变化,发现产卵后6种酶的活力均有下降。其中产卵前缠卵腺、副缠卵腺中乳酸脱氢酶活力分别为89、57 U/g,产卵后分别为56、45 U/g。产卵前缠卵腺、副缠卵腺中超氧化物歧化酶活力分别为78、47 U/mg产卵后缠卵腺、副缠卵腺中超氧化物歧化酶活力分别为51、32 U/mg。     

Cloning, characterization and expression of the LECT2 gene in grass carp

An expressed sequence tag of grass carp leukocyte cell–derived chemotaxin 2 (LECT2) gene was screened from an established intestinal cDNA library. Rapid amplification of cDNA ends gave rise to a full-length LECT2 cDNA (gcLECT2) with a complete open-reading frame of 474 bp, encoding 158 amino acids about 17.9 kDa. Homology search and sequence alignment showed that this deduced protein sequence shared a high identity with LECT2 from other vertebrates. Western blotting indicated immunological cross-reactivity occurs between grass carp and human LECT2 protein. This gcLECT2 genomic sequence is 1,868 bp in size, which consists of five exons and four introns. Real-time quantitative PCR analysis revealed that gcLECT2 gene is ubiquitously expressed in different tissues of healthy grass carp including brain, gut, liver, spleen, kidney, muscle and heart, while the expression levels were significantly increased in liver and spleen followed by Aeromonas salmonicida infection. 992 bp 5′-flanking region sequence was cloned and analyzed, where one CAAT box and one GC island were found. Our results showed that the LECT2 is suggested to be most possibly involved in the grass carp’s immune response.     

Molecular characterization and identification of facilitative glucose transporter 2 (GLUT2) and its expression and of the related glycometabolism enzymes in response to different starch levels in blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

Facilitative glucose transporters (GLUT) are transmembrane transporters involved in glucose transport across the plasma membrane. In this study, blunt snout bream GLUT2 gene was cloned, and its expression in various tissues and in liver in response to diets with different carbohydrate levels (17.1; 21.8; 26.4; 32.0; 36.3; and 41.9% of dry matter). Blunt snout bream GLUT2 was also characterized. A full-length cDNA fragment of 2577 bp was cloned, which contains a 5′-untranslated region (UTR) of 73 bp, a 3′-UTR of 992 bp, and an open reading frame of 1512 bp that encodes a polypeptide of 503 amino acids with predicted molecular mass of 55.046 kDa and theoretical isoelectric point was 7.52. The predicted GLUT2 protein has 12 transmembrane domains between amino acid residues at 7–29; 71–93; 106–123; 133–155; 168–190; 195–217; 282–301; 316–338; 345–367; 377–399; 412–434; and 438–460. Besides, the conservative structure domains located at 12–477 amino acids belong to the sugar porter family which is the major facilitator superfamily (MFS) of transporters. Blunt snout bream GLUT2 had the high degree of sequence identity to four GLUT2s from zebrafish, chicken, human, and mouse, with 91, 63, 57, and 54% identity, respectively. Quantitative real-time (qRT) PCR assays revealed that GLUT2 expression was high in the liver, intestine, and kidney; highest in the liver and was regulated by carbohydrate intake. Compared with the control group (17.1%), fed by 3 h with higher starch levels (32.0; 36.3; and 41.9%), increased plasma glucose levels and glycemic level went back to basal by 24 h after treatment. Furthermore, higher dietary starch levels significantly increase GLUT2, glucokinase (GK), and pyruvate kinase (PK) expression and concurrently decrease phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) mRNA levels (P?<?0.05), and these changes were also back to basal levels after 24 h of any dietary treatment. These results indicate that the blunt snout bream is able to regulate their ability to metabolize glucose by improving GLUT2, GK, and PK expression levels and decreasing PEPCK and G6P expression levels.