Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (Pseudosciaena crocea) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1: 100 with Ringer's solution and UV irradiated with doses ranging from 0-150 J cm (-2). The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm(-2), after fertilization, motility was < 10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm -2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 208 degrees C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures ( 28 degrees C, 38 degrees C or 48 degrees C) and five shock durations ( 4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 +/- 2.99%) were obtained at 38 degrees C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.

Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

Circadian rhythms in the growth and reproduction of the brown alga Undaria pinnatifida and gametogenesis under different photoperiods

Circadian growth rhythm of the juvenile sporophyte of the brown alga Undaria pirznatifida was measured with the computer-aided image analysis system in constant florescent white light under constant temperature (10 degrees C). The growth rhythm persisted for 4 d in constant light with a free-running period of 25. 6 h. Egg release from filamentous gametophytes pre-cultured in the light - dark regime was evaluated for six consecutive days at fixed time intervals in constant white light and 12 h light per day. Egg release rhythm persisted for 3 d in both regimes, indicating the endogenous nature. Temporal scale of egg release and gametogenesis in 18, 16, 12 and 8 h light per day were evaluated respectively using vegetatively propagated filamentous gametophytes. Egg release occurred 2 h after the onset of dark phase and peaked at midnight. Evaluation of the rates of oogonium formation, egg release or fertilization revealed no significant differences in four light-dark regimes, indicating; the great plasticity of sexual reproduction. No photoperiodic effect in gametogenesis in terms of oogonium formation and egg release was found, but fertilization in short days was significantly higher than in long days. Results of this investigation further confirmed the general occurrence of circadian rhythms in inter-tidal seaweed species.

Early development of germlings of Sargassum thunbergii (Fucales, Phaeophyta) under laboratory conditions

Morphology and culture studies on germlings of Sargassum thunbergii (Mertens et Roth) Kuntze were carried out under controlled laboratory conditions. Growth characteristics of these germlings grown under different temperatures (from 10 to 25A degrees C), irradiances (from 9 to 88 mu mol photons m(-2) s(-1)), and under blue and white light conditions are described. The development of embryonic germlings follows the classic "8 nuclei 1 egg" type described for Sargassaceae. Fertilized eggs spent 5-6 h developing into multicellular germlings with abundant rhizoids after fertilization. Under conditions of 20A degrees C, 44 mu mol photons m(-2) s(-1) and photoperiod of 12 h, young germlings with one or two leaflets reached 2-3 mm in length after 8 weeks. Temperature variations (10, 15, 20, 25A degrees C) under 88 mu mol photons m(-2) s(-1) significantly influenced the growth rate within the first week, although this effect became less obvious after 8 weeks, especially at 15 and 20A degrees C. Variation in germling growth was highly significant under different irradiances (9, 18, 44, 88 mu mol photons m(-2) s(-1)) at 25A degrees C. Low temperature (10A degrees C) reduced germling growth. Growth of germlings cultured under blue light was lower than in white light. Optimal growth of these germlings occurred at 25A degrees C and 44 mu mol photons m(-2) s(-1).

Early development of Costaria costata (C. Agardh) Saunders and cultivation trials

Costaria costata (C. Agardh) Saunders is one of common kelps distributed in many coastal areas worldwide; however, in China, no reports have been made on cultivation of the genus. To investigate potential cultivation of the species in the northern part of China, trials on isolation and preservation of the gametophytes were conducted using C. costata from Korea; growth and development of the gametophytes were observed. We showed that at 10 +/- 1A degrees C, 60 mu mol m(-2)s(-1) and 12:12 h (L:D), freshly released zoospores settled down within 1 hour, and then developed into the primary cell during the following 2 days. After a vegetative growth phase lasting 6-8 days, female gametophytes became 3-4 times larger in diameter than that of the primary cell, but still remained at a unicellular stage, while male gametophytes divided into 4-10 cells with only a slight change in size. Fertilization occurred within 10 days after the zoospores were released from the sporangia, and the apical and basal tissues of the juvenile sporophyte divided and differentiated into the blade and stipe. Temperature and irradiance influenced gametophytic vegetative growth and developmental patterns. Generally, low irradiance (15 mu mol m(-2)s(-1) and 30 mu mol m(-2)s(-1)) was unfavorable to the induction of fertility, but it enhanced female gametophyte division. The optimal conditions for vegetative growth were 15A degrees C and 30 mu mol m(-2)s(-1). After transplantation of the juvenile seedlings and after eight months cultivation, the harvested mature blade reached 194 cm in length and 32.7 cm in width. Our study proves that it is feasible to implement propagation and large scale cultivation of C. costata in northern China.

长江口海域富营养化的氮同位素特征研究

本论文以长江口海域2005年4个季度的现场调查为基础，测定了不同季节表层水体中悬浮颗粒有机物的δ15N值（δ15Np）及其它相关参数，讨论了不同季节长江口海域表层水体、长江河道及最大混浊带表层及底层水体、特征站位（上升流和非上升流区域）垂直水层水体中δ15Np分布及其与其他参数变化的相关性，综合分析了长江口富营养化水体陆源氮输入情况以及氮的生物地球化学变化，主要结果如下： 1.长江口海域表层水体中δ15Np值相对较低、分散程度相对较大，且不同季节δ15Np总体水平、离散程度不同。该海域农业化肥、大气沉降对该海域氮来源贡献可能相对较大，且可能具有较大程度的陆源氮输入变化、氮生物地球化学变化及氮分馏作用。 2.与枯水期相比，长江丰水期表层水体δ15Np较高，不同调查断面之间差别较小，悬浮颗粒有机氮（SPON）来源相对一致，且工业废水、生活污水的比重较大。枯水期、丰水期长江不同调查断面δ15Np和其他参数变化的相关性不明显，SPON生物地球化学作用导致的δ15Np分馏作用不明显；SPON来源及其δ15Np底值差异是长江水体δ15Np分布特征的主要影响因素。 3.总体上，和春、夏季相比，秋、冬季长江口海域表层水体中氮生物地球化学变化程度及其对氮分布、转化及悬浮颗粒有机氮分馏的影响较小；悬浮颗粒有机氮分解反应的影响范围在秋季最大，而同化作用比重在冬季最大。 4.长江口近岸海域悬浮颗粒有机氮分解作用对氮分布、转化及悬浮颗粒有机氮分馏的影响总体较大。该影响作用夏季最大，冬季最小，在最大混浊带海域较大而在河道水域较小。远岸海域同化作用及分解作用的影响程度接近。其中分解作用的影响在秋季最大，春、夏季次之，冬季最小；冬季同化作用的影响最大，秋季最小。远岸海域南部、北部水域同化作用程度及其影响均较大，中部海域及靠近陆地的东部海域分解作用程度及其影响较大。 5.长江河道及最大混浊带海域陆源氮输入来源、氮生物地球化学变化及其分馏作用在不同水层、不同站位之间的差别春季最大。和底层相比，长江河道表层水体陆源输入氮来源、氮生物地球化学变化及其分馏作用在不同季节、不同站位之间的差别较大，而最大混浊带表层水体中该差别较小。悬浮颗粒有机氮分解作用和同化作用对长江河道表层水体中氮分布、转化及悬浮颗粒有机氮分馏的影响接近，底层水体中分解作用影响较大。最大混浊带海域情况相对复杂。 6.与非上升流水域相比，上升流水域垂直水体中δ15Np分布季节差异大，台湾暖流对其影响较大，尤其是秋季，导致其垂直水体δ15Np较低。近岸的17、41号水域垂直水体中悬浮颗粒有机氮生物地球化学变化对其垂直水体中氮分布、转化及悬浮颗粒有机氮分馏作用的影响较明显。

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

大西洋庸鲽Hippoglossus hippoglossus和大西洋鲑Salmo salar配子质量研究

种质问题是养殖健康发展的基础。在鱼类养殖中，卵子和精子的质量直接关系到受精、胚胎发育，仔稚鱼发育以及幼鱼生长等一系列过程。本论文针对大西洋庸鲽和大西洋鲑的配子质量进行研究。研究内容涉及大西洋庸鲽精子冷冻保存方法；促性腺激素释放激素类似物（GnRHa）使用对其精子冷冻保存效果、以及脂肪酸组成的影响；野生和驯养大西洋鲑卵子在脂肪酸、类胡萝卜素、矿物盐方面的差异比较。 精子冷冻保存通过提高对精子的利用效率，进而对于种质改良，推进鱼类养殖科研和生产具有重要意义。本实验建立了大西洋庸鲽精子大容量冷冻保存方法。八种抗冻剂冷冻保存实验结果表明：10% 及15% DMSO配以 HBSS 或KS 的抗冻剂组合冷冻保存效果最佳，4 mL体积冷冻保存可获得与1.6 mL同样的保存效果。 在繁殖季节后期注射GnRHa激素缓释剂，可获得质量稳定的大西洋庸鲽精液，将激素注射方法与精子冷冻保存方法相结合对于提高雄鱼利用率，扩大生产规模具有重要实用价值。本项研究分三个时间采集注射GnRHa激素后的雄鱼精子以及同期未注射激素的雄鱼精子，对所有精子样品使用同样的方法进行冷冻保存，检测冷冻保存后解冻精子的受精率与活力。结果表明，激素注射与否对于冷冻保存后精子的受精率和活力无显著影响，两类冷冻精液均达到鲜精水平。实验结果还表明，注射激素14天后的精子的密度显著的降低。说明GnRHa激素的使用可以显著降低精子密度，但不会影响精子的冷冻保存效果。 本相研究同时对注射GnRHa 缓释激素和未注射GnRHa 缓释激素的大西洋庸鲽精液脂肪酸成分进行分析，以检测该激素使用对精子生化组分的影响。结果表明激素的使用对在DHA (22:6n-3,二十二碳六烯酸)、EPA(20:5n-3,二十碳五烯酸)、AA(20:4n-6，花生四烯酸)等重要脂肪酸，不饱和脂肪酸、饱和脂肪酸以及n-3、n-6等重要种类的脂肪酸总量及其比例没有显著影响。精液脂肪酸中DHA含量最高，约占25%；PUFA约为44%。 作为世界性的重要养殖品种，野生和驯养大西洋鲑在形态、生化组成以及遗传 等方面表现出的差异被广泛关注。本论文，对野生和驯养大西洋鲑受精卵关键生化成分进行分析，通过与野生受精卵比较阐明驯养受精卵的质量状况，为亲鱼营养需求提供指导依据。本实验中野生配子和驯养配子的受精率没有显著差异，但重要脂肪酸组成、类胡萝卜素以及矿物盐含量都存在多方面显著差异。两类受精卵脂肪酸中含量最高的依次为18:1n-9（油酸）、DHA（二十二碳六烯酸）、16:0（棕榈酸）、EPA（二十碳五烯酸）。野生受精卵的单不饱和脂肪酸总量显著高于驯养受精卵，而多不饱和脂肪酸（PUFA）比例显著低于驯养的受精卵。在主要必需不饱和脂肪酸（EFA）中，DHA和EPA在野生受精卵中的比例高于驯养受精卵，AA（花生四烯酸）低于驯养受精卵。野生受精卵虾青素（Ax）的含量低于驯养受精卵而鸡油菌素（Cx）含量高于驯养受精卵。野生受精卵中多种矿物盐的含量（铝、铜、铁、硒和锌）含量显著高于驯养的受精卵。差别最大的为铜。诸多方面的差异表明，野生亲鱼与驯养亲鱼产出的卵子确实存在显著差异，因此关注亲鱼的营养极为重要。

红毛菜发育过程及其生理基础

红毛菜（Bangia Lyngb.）属于红藻门，与紫菜属同属红毛菜科，其味道和营养都优于紫菜。目前红毛菜栽培产业已在我国福建莆田展开，但栽培技术还有待提高。海藻栽培技术的发展和成熟依赖于对其生长发育过程的认识。本研究针对红毛菜发育过程及相关光合生理展开，并初步探讨了一采自山西娘子关泉淡水红毛菜群体（FWB）的系统地位。 色素突变标记的壳孢子萌发特征表明最初两次分裂产生的4细胞决定了完整植株的形态建成。成熟植株，为雌雄异体。雌性生殖器果胞的标志性分化结构为原始受精丝，环境因子是促发原始受精丝发展的外部因素，其膨大程度随受精的延迟而增大。原孢子是主要的无性生殖孢子类型，在不良环境中，藻体也会形成内生孢子或休眠包囊，或者藻体断裂后重新形成完整的植株。 红毛菜的生长发育很大程度上受环境因子的控制。高温不利于配子体的发育，15-20 ºC比较适宜。红毛菜无性繁殖的最适温度-光照组合为20 ºC-8 h，有性繁殖为15 ºC-12h。 不同发育阶段，PSII实际光合效率（Y(II)）与细胞的健康状况以及光合器官完整性及其在细胞内的分布有关，而与细胞的类型关系不大。健康的假根细胞、已分化未成熟的精子以及果孢子细胞均具有很高的Y(II)。色素体由中间位变为围周位，中央大液泡（营养藻丝）和大小纤维囊泡（成熟孢子与精子）的产生，使得细胞Y(II)降低。刚放散的壳孢子Y(II)很低，说明在壳孢子由贝壳基质释放到自由水体过程，光合作用受到一定程度抑制；而2h后，Y(II)开始恢复，rbcL的转录水平非常高，为孢子的萌发储备物质和能量需求。 在失水和低盐胁迫下，藻体均维持较高的Y(II)。干出处理至藻体重量不再变化，复水后Y(II)可回复初始水平。海生红毛菜在100％淡水培养基中（约20ºC）培养7天后，部分雄性藻体依然活着。从而体现了红毛菜位居高潮带的生理优势。 FWB终生行无性繁殖，藻体形态与发生以及染色体数目（4条）与海生群体没有区别。而rbcL-rbcS Spacer序列显示，红毛菜海生群体（无性和有性）具有完全相同的序列，而FWB与它们有5bp差异，但是与欧洲、北美地区的淡水群体仅1bp不同，初步说明所有淡水红毛菜群体具有共同的原始起源。

长江口海域氮的同位素特征及其环境意义

本论文以稳定氮同位素（δ15N）技术为基础，对长江口海域氮的同位素特征及其环境意义进行了研究。确立了一套完整的水体中氮的稳定同位素分析预处理方法，并运用该方法对2006年长江干流以及长江口海域表层水体中的溶解态硝酸盐和悬浮颗粒物的δ15N特征进行分析，根据不同季节、不同区域内其δ15N值的变化研究水体中氮的迁移、转化等生物地球化学过程，揭示其环境行为，从而对该海域的氮循环机制进行探索。主要结果如下： 稳定氮同位素的分析准确与否，预处理过程是关键。不同形态、不同水体中氮的预处理方法不同。本文以传统的蒸馏法为基础，对适合海水中溶解态硝酸盐的氮同位素分析预处理方法进行研究并改进，并进行了不同实验条件的验证，效果较好。此外，对淡水体系中溶解态硝酸盐的氮同位素分析预处理方法以及悬浮颗粒物的氮同位素分析方法也进行了研究，确立了一套完整的水体中氮的稳定同位素分析方法，对δ15N技术在河口氮循环研究中的广泛应用提供了基础。 依据上述方法，对2006年2、5、8、11月份长江口海域表层水体中的溶解态硝酸盐δ15N值（δ15N-NO3-）进行分析。研究发现，δ15N-NO3-分布范围在0.4‰到6.5‰之间，平均为3.5‰，具有明显的时空分布特点，在不同季节不同区域内所受的物理、生物地球化学作用不同。口门内，δ15N-NO3-的季节变化主要受长江径流输入影响，来源单一。最大浑浊带，δ15N-NO3-的分布规律不明显，保守混合行为较差，说明该区域的影响作用复杂，不同季节水体中发生的生物地球化学作用存在差异。外海区，δ15N-NO3-的季节变化明显，春季>秋季>夏季>冬季，与生物对硝酸盐的吸收程度变化相一致，反映了生物作用在该区域的影响显著。 同样在2006年2、5、8、11月份本文对长江口海域表层水体中悬浮颗粒物的δ15N（δ15Np）组成进行了研究。δ15Np值分布在0.6-8.2‰之间，同样具有明显的时空分布特点，其变化趋势与陆源输入和水体中氮的生物地球化学过程有关。口门内，表层水体中δ15Np的变化主要受长江径流的陆源输入影响，生物地球化学作用影响较弱；最大浑浊带，水体中的悬浮颗粒有机氮受微生物的降解活动影响明显，各季节均存在不同程度的颗粒物分解作用；外海区，陆源输入减弱，悬浮颗粒物的δ15Np值主要受微藻的同化吸收作用以及一定程度的颗粒物分解作用影响。 长江口海域水体中溶解态硝酸盐和悬浮颗粒有机氮之间存在重要的相互转化作用，二者之间δ15N的变化及其相互关系反映了一定的生物地球化学变化和环境信息。总体而言，长江口海域表层水体中δ15N-NO3-的分布水平较δ15Np略低，二者之间的分馏ε总体偏正。其中，δ15N-NO3-与δ15Np的最低值均出现在2月份，说明该季节水体溶解态硝酸盐和悬浮颗粒物都可能受到一致的外源输入影响，其内部生物地球化学作用较弱。5月份和11月份水体中的δ15N-NO3-值和δ15Np值均各自水平相近，说明在这两个季节溶解态硝酸盐和悬浮颗粒物都可能受到程度相近的外源输入或生物吸收作用的影响，分馏ε偏负程度较大。8月份δ15N-NO3-值较低而δ15Np值较高，分馏ε在四个季节中最大，可能是该季节程度较大的颗粒物分解作用影响所致。 2006年长江干流表层水体中溶解态硝酸盐及悬浮颗粒物的δ15N组成也具有明显的时空分布特点，二者季节变化规律相近，丰水期（5、8月份）δ15N值较高，而枯水期（2、11月份）较低。δ15N-NO3-与δ15Np之间呈现明显的正向相关关系，其空间分布趋势相同，自长江上游至下游δ15N值逐渐升高，说明二者均受到相似的氮来源影响；长江上游氮的来源均以大气沉降和农业源（无机化肥和土壤有机氮）为主，而中、下游水体氮的来源则都偏重于工业及生活排污的贡献，随着人类活动程度以及工业化、城市化程度的升高，硝酸盐及悬浮颗粒有机氮的来源发生变化，其δ15N值相应增加。

LABORATORY HYBRIDIZATION BETWEEN CRASSOSTREA ARIAKENSIS AND C. SIKAMEA

TO understand possible reproductive interaction between Crassostrea ariakensis (Fujita, 1913) and C. sikamea (Amemiya, 1928), which coexist ill estuaries of China and Japan, we conducted 2 X 2 factorial crosses between the two species. Asymmetry in fertilization success was observed where C. sikamea eggs can be fertilized be C. ariakensis the receprocal cross resulted in no fertilization. Fertilization Success ill C.sikamea female X C. ariakemvis male (SA) crosses was lower than that in the two intraspecific crosses and produced larvae that had similar growth the rate as their maternal species during the first nine days because of maternal effects. After that, genome incompatibility casted negative effects on the growth and survival of the hybrid larvae. Most hybrid larvae died during metamorphosis. but a small number of spat survived. Genetic analysis revealed that the survived SA spat contained DNA from both species and were the hybried. This study demonstrates that hybridization between C. ariakensis and C. sikamea is possible in one direction.

Temperature influence on the spawning performance of artificially-matured Japanese eel, Anguilla japonica, in captivity

We studied the influence of temperature on the spawning performance of artificially matured Japanese eels, Anguilla japonica, in captivity. We used routine hormone injections to bring females and males to maturity in separate aquaria. We recorded the behavior of three pairs of such hormone-treated matured eels in an aquarium (2 replicates) at four temperatures: 14, 18, 22, and 27 degrees C, respectively. They became active and frequently left the bottom swimming in the water column, and spawning events occurred. Females released eggs in the water column around the activity peaks. Males preceded females in reaching activity peaks (presumably the timing of sperm ejection and egg release), possibly resulting in the low fertilization we observed in this experiment. Males and females returned back to the aquarium bottoms and became quiet after spawning. On several occasions, male-female or female-female pairs were observed to 'cruise together' in the water column for several to tens of seconds prior to egg releasing, but no courtship behavior indicative of spawning such as pairing and chasing was observed in the eels in our study. Our results suggest that 18-22 degrees C might be the thermal preference for spawning for Japanese eels, which approximates the temperature range of the 500 m deep water layer around the Mariana Islands seamount area, the presumed spawning site for the Japanese eel.

Experimental evaluation of heterobeltiosis and heterosis between two populations of Pacific abalone Haliotis discus hannai Ino

A2 x 2 factorial cross between two populations of Pacific abalone Haliotis discus hannai Ino, collected separately from Dalian (D) in China and Miyagi (M) in Japan, was conducted to compare performances in fertilization rate, hatching rate, metamorphosis rate and growth at days 20, 43, 160 and 330 between purebreds (DD and MM) and crossbreds (DM and MD) and investigate the magnitude of heterobeltiosis (better parent) and heterosis (mid-parent). Heterobeltiosis and heterosis for all the traits analyzed were evidently different between crossbreds DM and MD. Heterobeltiosis in the crossbred DM varied among traits, with values of 2.5% for the fertilization rate, 2.2% for the hatching rate, -1.9% for the metamorphosis rate and 7.4% for the growth at the (lay 330. The crossbred DM displayed positive heterotic values for fertilization rate (5.4%), hatching rate (7.4%), metamorphosis rate (7.6%) and growth (12.0%) at the day 330. However, both heterobeltiosis and heterosis for all the traits in the crossbred MD were negative except those for the growth at days 20 and 43. The results indicate the importance of selecting superior hybrid varieties if the exploitation of hybrid vigor is considered in the Pacific abalone breeding program.

Genetic parameter estimates for growth traits at early stage of Pacific abalone, Haliotis discus hannai Ino

Heritability and genetic and phenotypic correlations were estimated for juvenile growth traits of Pacific abalone Haliotis discus hannai Ino. The estimates were calculated from shell length and shell width measurements on progeny resulting from 12 half-sib families and 36 full-sib families obtained using artificial fertilization of mating three females to each male. The measurements were taken at 10, 20 and 30 d after fertilization. It was found that heritability estimates based on sire component ranged from 0.23 to 0.36 for shell length and 0.21 to 0.32 for shell width. Heritability estimates from dam component were larger than those from sire component at three ages, indicating presence of maternal effects, non-additive genetic effects and common environmental effects. Phenotypic correlations were significant at three ages (P < 0.05), with values of 0.92, 0.93 and 0.92, respectively. Genetic correlations from the paternal half-sib correlation analysis were highly positive at three ages, with values of 0.50, 0.78 and 0.81, respectively. The results suggest that selective breeding is an effective approach to improving growth traits of Pacific abalone stocks.

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabrearn (Pagrus major). Mean (+/- S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0 +/- 5.4, 92.8 +/- 1.9, and 91.8 +/- 5.2%, respectively; for fresh sperm, they were 87.5 +/- 7.7, 95.8 +/- 2.4, and 93.8 +/- 4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P < 0.05), there was no effect (P > 0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8 +/- 5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0 +/- 7.2% had normal morphology, 20.6 +/- 3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4 +/- 4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased. (c) 2007 Elsevier Inc. All rights reserved.