【作者】 郝振林；

【导师】 张秀梅；

【作者基本信息】 中国海洋大学 ， 增殖养殖工程， 2010， 博士

【摘要】 本文以金乌贼为研究对象,采用实验生态学、组织学和生理学手段,研究了金乌贼亲体培育及人控环境下的繁殖行为；性腺发育和卵子发生的组织学；幼体金乌贼的生长发育及消化酶活性的变化；视觉发育；环境条件的变化对幼体金乌贼摄食行为的影响以及利用荧光染色剂(ALC)标志金乌贼内壳的放流标志技术。主要研究结果如下：1.以采自日照岚山头金乌贼为研究对象,观察了养殖池内金乌贼亲体的交配和产卵行为,比较了不同产卵基附卵效果的差异,分析了金乌贼胴长、体重与怀卵量之间的关系,确定了金乌贼亲体的产卵节律。结果显示：亲体金乌贼求偶、追偶、交配、产卵、扎卵等行为明显；不同产卵基附卵效果差异显著,网绳产卵基和柽柳产卵基附卵效果较好,地肤草较差；金乌贼怀卵量与胴长和体重之间相关关系不显著,怀卵量浮动于500～1500粒之间,平均怀卵量为1114粒；产卵时间较有规律,主要集中于21时至次日凌晨3时。2.以采自日照岚山头金乌贼亲体5尾(3雌,2雄)和金乌贼幼体39尾为研究对象,研究了亲体金乌贼生殖系统的结构特性以及金乌贼幼体发生性腺分化的时间。结果表明,金乌贼属雌雄异体。雄性生殖系统包括精巢、输精管、精囊、副性腺和精荚囊。精荚囊中充满了排列紧密的成熟精荚。精荚为棒球棒状,由体腔中的精子、接合体、螺旋发射器和帽子组成。精荚纵切面显示精子以“V”字型储藏于精荚体腔内,横切面显示精子呈螺旋状储藏于精荚体腔内,且在接近体腔中心位置螺旋状尤为明显。雌性生殖系统包括卵巢、输卵管、输卵管腺以及附属腺。幼体形成卵巢的时间约为孵化后7d,此时显微镜下可观察到卵巢中包含一些着色较浅的卵母细胞。根据卵母细胞的大小、卵母细胞核的形态及卵母细胞与滤泡细胞的结构变化,将金乌贼卵子发生划分为8个时期。3.对自然海区及养殖池内采集的金乌贼受精卵进行了人工孵化,对孵化后金乌贼幼体生长发育的生物学指标进行了测量,同时分析了酸性蛋白酶、脂肪酶等消化酶随日龄的活性变化。结果显示：受精卵孵化率受水温影响差异不显著,但受盐度影响差异显著；小水体条件下金乌贼幼体的存活率偏低为81.4%,大水体条件下,存活率较高为100%；如初期饵料充足,幼体乌贼规格整齐,放养密度较低时不会出现自残现象；金乌贼幼体生长迅速,属异速生长型；胴宽朋同长、壳宽/壳长的比值随生长呈下降趋势；此外,金乌贼幼体的酸性蛋白酶和脂肪酶活性均随日龄的增加而升高,至孵化后210d,分别达158.3 U/mL和183.2 U/mL。4.运用显微和超显微方法对金乌贼的视觉发育进行了研究。结果表明：金乌贼视网膜内四层结构清晰可见,由外向内依次为感杆层、次级感杆层、内界层、神经纤维层。1)感杆层,感杆层由许多排列规则、紧密的顶端突起组成,顶端突起外附有微绒毛。纵切面观察发现,微绒毛呈一定角度排列于每一个顶端突起周围,但顶端却未见微绒毛；横切面观察发现,顶端突起形状多样,呈圆形、纺锤形、三角形等不同形态。微绒毛呈放射状排列在顶端突起周围,但有些部位缺少微绒毛。视网膜发育早期阶段,微绒毛较短,且排列规律性欠佳,随生长发育,微绒毛逐渐变长,排列较有规律。此外,顶端突起中含有色素颗粒和小泡,小泡源于微绒毛,微绒毛失去小泡时便会萎缩。小泡内含有色素颗粒,当到达顶端突起时便会破裂并将色素颗粒释放出来。随着色素颗粒的增多,色素颗粒常会在顶端突起底部聚集,形成集合体。2)次级感杆层,次级感杆层中细胞质含有色素颗粒、小泡、线粒体、高尔基复合体和髓样体。线粒体和高尔基复合体数量随生长发育逐渐增多,髓样体规格和数量也随生长不断增加。随生长发育次级感杆层中细胞核不断穿越极板进入内界层,进入内界层的细胞核含有较多异染色质,形状变得细长且电子亮度加深。支持细胞数量随生长发育逐渐增多,其微绒毛嵌入感杆层。3)内界层,内界层中富含线粒体、核糖体、高尔基复合体、内质网和髓样体,且数量随生长发育明显增多。4)神经纤维层,神经纤维层内含有感受细胞轴突、神经纤维、线粒体、高尔基复合体和神经胶质细胞。神经纤维层的机能随生长逐渐趋于完善。另外,测量计算得知,感杆层厚度及单位面积微绒毛表面积的总量随生长与日龄呈幂函数关系,晶状体直径随生长与日龄呈线性关系。总结发现,金乌贼的视敏度随生长发育不断增加。5.研究了不同底质(玻璃底、沙底),光照度(2501x、O.11x),视角(视野前方、上方、下方)对金乌贼摄食行为的影响。结果表明：不同底质条件下,金乌贼的摄食行为表现出显著差异,沙底条件下,金乌贼表现出明显的隐蔽行为；金乌贼具有较高的视敏度,在光照强度2501x与0.11x条件下摄食行为差异不显著；金乌贼摄食视野上方食饵的成功率较摄食视野下方或前方时低约20%；在光照度2501x条件下,胴背长43mm、48mm、56mm的金乌贼对食饵反应距离分别为：130.0cm、135.5cm、143.1cm,光照度降至0.11x时,分别为：115.0 cm、120.6 cm、126.3 cm.6.以金乌贼为实验对象,利用荧光物质一茜素络合指示剂(Alizarin Complexone ALC)浸泡金乌贼幼体,对其内壳进行标志,所用荧光染色剂浓度为6.0～8.0×10-3%,浸泡染色时间24h。将标志组和对照组的金乌贼幼体分别暂养于1.5m×1.5m×1.5m水泥池30d,后将暂养的幼体放养于面积为2667m2的室外土池,并在60d后,将平均胴背长达91.4mm(对照组)、87.6mm(标志组)金乌贼幼体移入室内水泥池越冬,整个实验历时210d。实验期间,分别在标志后15、30、45、60、90、210d对金乌贼进行随机取样,解剖出内壳观察标志色保留状况并对金乌贼生长发育及存活率进行测量。结果显示,标志金乌贼的成活率为100%；方差分析显示,标志组和对照组金乌贼的生长发育差异不显著(P>0.05)；210d后内壳骨针部仍清晰保留初染时的半椭圆形淡紫色圆圈；本实验所采用的ALC内壳标志方法操作容易,可一次性大量进行标志处理,鉴别简单,无需借助其它仪器,肉眼便可直接观察到标志色,并且标志色保持率高,保留时间长,是一种理想的金乌贼放流标志方法。更多还原

【Abstract】 This paper reviewed the recent literatures on the biology and enhancement technique of Sepia esculenta, including morphology, fishery biology, behavior ecology, and genetics. The domestication and propagation behaviour in artificial environment, the histology observations on gonad and oogenesis, the growth and digestive enzymes, the development of retina, the feeding behavior in different environmental factors, the fluorescent marking technology were studied in this paper. The most results are summarized as follow.1 Studies on the domestication and the propagation behaviour in cuttlefish, Sepia esculenta (Cephalopoda:Sepiidae)S.esculenta were collected from Rizhao Lanshan as researchful object in this paper. The matching and reproduction behaviour of mature S. esculenta were observed in breeding pond, the effect of different spawning substrate were studied, the relationship between the mantle length and body weight of S.esculenta and fecundity was analyzed, the spawning rhythm of mature S.esculenta was founded. The investigation results showed that the courtship behaviour, chasing behaviour, matching behaviour, spawning behaviour were obviously observed in the process of mating; there were significantly differences-between different spawning substrate, compared with the Kochia scoparia substrate, The fishing net substrate and Tamarix chinensis substrate are better; there were no founction relation between the fecundity and he mantle length and body weight of S.esculenta, the fecundity was mostly between 500 and 1500, and the average was 1114;the spawning time was very disciplinarian and mostly between 21:00 and the next day 03:00.2 Histology observations on gonad and oogenesis in cuttlefish, Sepia esculenta (Cephalopoda:Sepiidae)Sepia esculenta, is a most commercially important species of coastal fishes in China and Japan,however, little is known about the mature reproductive system, the gonadal development and the oogenesis of cultured juveniles. To determine the histological character of the mature reproductive system and the juveniles size at gonadal differentiation, five adults (3 females,2 males) cuttlefish and 39 cultured juveniles ranging from 1 to 88 d old after hatching are used in this experiment. The results show that S.esculenta is gonochorism and the genital system of the males consists of spermary, spermaduct, vesicular seminalis, prostate gland and Needham’s Sac.The Needham’s Sac is completely filled with tightly packed and well developed spermatophores. The spermatophore is shaped like a baseball bat and consists of an elongated sperm mass, a cement body, a coiled spring-like ejaculatory organ, and a cap. Sperm mass are "V"-like stored in the cavity of spermatophore from longitudinal section. From the transverse section, they are seen spirally stored in the spermatophore especially near to the centre. The genital system of the females consists of ovary, oviduct, albumen gland and affiliated gland.The time that gonads are differentiated into ovaries in juveniles is at about 7th d after hatching when the gonads contained some staining gonocytes. Eight stages of oocytes were indicated basing upon the degree of formation and development of the oocytes and associated follicle cells via the histological observation.3 Studies on growth and digestive enzymes of juvenile cuttlefish, Sepia esculenta (Cephalopoda:Sepiidae)The S.esculenta were collected from Rizhao Lanshan as researchful object in this paper, the oosperms of natural collection and manul collection were hatched under the artificial condition, the hatchlings were reared with superfluous food everyday, the developmental characteristic of S.esculenta was studied and the relation between age and developmental parametey was analyzed.The investigation results showed that the temperature significantly affected the oosperm hatching time; the oosperm hatching rate was not significantly affected by temperature, but significantly affected by salinity; the survival rate of S.esculenta was 81.4% in small water, and 100% in big water; the autotomy was not appearance when the foods were eough, the size of S.esculenta was in order and the density of S.esculenta was low; the hatchlings grow rapidly with the development. The acid protease activity and the activity of lipase of S.esculenta were increasing with ages, which werel58.3 U/mL and 183.2 U/mL respectively when they were 210d old after hatching. 4 Development of the retina in the cuttlefish, Sepia esculenta (Cephalopoda: Sepiidae)To provide morphological bases for functional studies, the development of the photoreceptor cells in the retina of a cuttlefish, Sepia esculenta from newly hatched juveniles to the adults, was examined by light-and electron-microscopy. Four layers can be distinguished in the retina according to the observation.1),rhabdomeric layer. Which increases in thickness gradually during the development. On the lateral surface of the growing apical processes in the rhabdomeric layer, microvilli increase in length and regularity.In longitudinal sections of the apical processes, the microvilli are arranged at right angles to the long axis of each apical process, but the top of the apical process is often free from microvilli. In cross-sections, the microvilli arise radially in all directions from the round circumference of the apical process. Usually the pigment granules coming from microvilli are crowded near the distal end and base of the process, gradually increase in number, often form a large aggregation. Some microvilli start to decay as more and more vesicles coming from microvilli. 2), sub-rhabdomeric layer. There exist numerous pigment granules, a few mitochondria, sparsely scattered small vesicles of various shapes, golgi apparatuses, and myeloid bodies. At first they are very few and small, and then increase in number,In the eyes of newly hatched juveniles, the number of the nucleus in the sub-rhabdomeric layer is very large, but decreases much with the development as they ceaselessly pass through the basal lamina into the inner-segment. The supporting cells increase in number and send out very long microvilli into the rhabdomeric layer, the electron-lucent of the whole the sub-rhabdomeric layer is increasing.3), inner segmental layer. They are rich in membranous organelles. The cytoplasm contains microtubules and numerous free ribosomes. Mitochondria increase in number remarkably.4),plexiform layer. Which consists of the receptor cell axons, nerve fibers, mitochondria, golgi apparatuses, and the glial cells. With the development of the retina, the structure of plexiform layer become stronger. Morphometric data on the rhabdomeric microvilli from the newly hatched to the adult retinas are presented. The thickness of the rhabdomeric layer and the total surface area of the rhabdomeric microvilli present beneath a unit surface area of the retina show a logarithmic increase with developmental days, the diameter of lens shows a linear increase with days. The results of our research show that the visual acuity and sensitivity of S.esculenta continuously increased with the development.5 Effects of different environmental factors on feeding behavior of juvenile cuttlefish, Sepia esculenta (Cephalopoda:Sepiidae)This paper investigated the effects of substrate(glass substrate,sand substrate),visual angle(frontage,above,below) on feeding behavior of sepia escul-enta. The results in the present study indicated that substrate significantly affected the feeding behavior of Sepia esculenta,and covert behavior could easily be seen in the process of Sepia esculenta’s feeding behavior in sand substrate. There were no significant differences between 2501x and 0.11x; the chance of successfully preying the shrimp which is above the view is about 20% lower than o-ther bearings; the reaction distance of Sepia esculenta(doral mantle length, DML (43mm、48mm、56mm))is 130.0cm、135.5cm、143.1cm respectively in 2501x;and 115.0cm、120.6cm、126.3cm in 0.11x.6 The study of fluorescent marking technology in cuttlefish Sepia esculenta (Cephalopoda:Sepiidae)It is very important to establish a kind of simple and useful marking method to evaluate multiplication and releasing effect and the current situation of fisheries resource. The golden cuttlefish were chosen to be the study matieral in this article. The larval golden cuttlefish hached after 15d were immersed in 6.0～8.0×10-3% alizarin complexone(ALC)for 24h. The tagged group and control group were reared in 1.5m×1.5m×1.5m indoor pond for 30d, and then they were removed to 2667m2 outdoor pond. After 60d, their mean doral mantle length grew to 91.4mm (control group) and 87.6mm (tagged group) respectively, they were removed to indoor pond for over winter. During 210d experiment, the golden cuttlefish were sampled randomly in order to examine the growth and the dye-retention of the tagged cuttlebone after tagged 15、30、45、60、90、210d. The results of the experiment showed that this marking method is very effective, and the survival rate of the tagged golden cuttlefish is 100%. There are no significant differences between the growth of the tagged group and the control group (P>0.05). The half tagged lavender ring is very clear in the cuttlebone even after tagged 210d. Additionally, according to this method, we could tag lots of samples easily in one time, and the retention of marker can last long period. Due to the maneuverability and easy to distinguish tagged ones, it is suggested that this method could be a good marking method for golden cuttlefish.更多还原