3.2.3 LARVAL ASSAYSE. chloroticus l

3.2.3 LARVAL ASSAYS
E. chloroticus larvae were raised for 28 d in a standing-renewal system composed of 2 L glass jars placed in a flowing seawater bath to maintain a near constant temperature. Water temperature was 16° C ±1 throughout the whole experiment and pH was measured in each jar at one and three week post-fertilisation. Jars were gently stirred by a system of motorised paddles as described in Strathmann (1987), to provide adequate oxygenation. Embryos were placed in their culture jar within 1 h post-fertilisation at a density of 1 embryo/ml. All glassware was washed with hot water, soaked in 10% hydrochloric acid for a minimum of 4 h, rinsed and soaked in distilled water for 12 h and rinsed in FSW before use. FSW in jars was renewed three times per week by inverse siphoning. From 4 d post-fertilisation, larvae were fed following each water change with Dunaliella tertiolecta at a concentration of 8000 cells/ml of culture. Dunaliella stocks were rinsed thoroughly by three successive centrifugations (1201 g for 5 min) and resuspensions in clean FSW before being fed to the larvae, to remove traces of algal culture medium that was rich in essential metals.
All jars were sampled at 4 d and 11 d post-fertilisation; jars in the Early timing group were sampled at 25 d, and jars in the Late timing group at 13 d and 27 d postfertilisation. During sampling, the water level in each jar was lowered to 750 ml to increase larval density and 3 x 15 ml aliquots were taken per jar. Jars were randomly numbered and all measurements were done blind with regard to the treatment classification. Number of live larvae was measured in each aliquot using a dissecting microscope (40x magnification). Each live larva was assigned to one of eight developmental categories (Table 3.2). The first ten normal larvae encountered were preserved in 40% ethanol to be photographed under a compound microscope (50x magnification). When two developmental stages were considered normal (e.g. at 25 d post-fertilisation both 6-armed plutei and 8-armed plutei were rated as normal), only larvae belonging to the most advanced developmental stage (e.g. 8-armed plutei at 25 d) were taken for morphometric measurements. Measurements of larval body and arm lengths (average of R and L arms where possible; Table 3.2) as well as presence and size of the rudiment in the late larval stage were done using the software ImageJ.

結果 (中文) 1:

3.2.3 幼虫化验E.chloroticus 幼虫对 28 d 提出了在站更新体系组成的 2 L 玻璃瓶放置在流动的海水浴保持近乎恒定的温度。水温度为 16 ° C ± 1 贯穿了整个实验过程和 ph 值衡量每个在一至三周后受精的罐子里。罐子里轻轻地感动了机动桨系统中所述 Strathmann （1987 年），以提供足够的氧。胚胎被放置在其文化罐内的 1ml 胚胎在密度 1 h 后受精。所有的玻璃器皿是用热水洗净、 4 h 至少 10%盐酸溶液中浸泡、 冲洗和浸泡在蒸馏水中为 12 h 和使用前搅拌摩擦焊在漂洗。搅拌摩擦焊在罐子里被更新三次，每周由反虹吸现象的发生。从 4 d 后受精，幼虫喂食后有杜氏盐藻藻粉毫升的培养液 8000 细胞浓度在每个水更改。彻底送入到幼虫，以去掉了丰富的基本金属的藻培养基之前将杜氏盐藻股票冲洗由三个连续洗涤 (1201 g 为 5 分钟) 和 resuspensions 在干净的搅拌摩擦焊。所有罐子都取样 4 d 和 11 d 后受精;早期的定时组的震击器取样 25 d 和后期的定时组在 13 d 和 27 d postfertilisation 的罐子。在取样，过程中每个瓶子里的水位降至 750 毫升增加幼虫密度和 3 x 15 毫升被每罐。所有测量都进行盲关于治疗分类和罐子被随机编号。活幼虫数量被衡量每个分装用解剖显微镜 （40 x 放大）。每个活幼虫被分配到八个发展类别 (表 3.2) 之一。遇到的第一次十正常幼虫完好以 40%的乙醇，拍摄下复合显微镜 （放大 50 倍）。当两个发展阶段被认为是正常 （例如在 25 d 后受精 6 武装 plutei 和武装 8 plutei 被评为为正常），只幼虫属于最先进的发展阶段 (例如 8 武装在 25 d plutei) 来进行形态学测量。幼虫的身体和手臂长度测量 （平均的 R 和 L 武器在可能的情况下;表 3.2），以及进行的存在和大小的雏形在幼虫阶段末期使用软件 ImageJ。

結果 (中文) 2:

3.2.3幼虫ASSAYS
E. chloroticus幼虫在放置在流动海水洗澡，以保持接近恒温2升玻璃瓶组成的常设更新系统提高了28天。水温为16℃±1在整个实验和pH值在每个罐子在一个和三个后一周受精测定。如在Strathmann（1987）描述的，以提供足够的氧合罐子轻轻机动桨叶系统搅拌。胚胎被以1胚胎/ ml的密度置于它们的培养罐1小时后受精内。所有玻璃器皿洗涤用热水，浸泡在10％盐酸最少4小时，清洗，并在蒸馏水中浸泡12小时，使用前在FSW漂洗。FSW在罐子被反虹吸更新，每周三次。从4天受精后，幼虫饲喂继8000个细胞/ ml培养的浓度杜氏tertiolecta每个水的变化。杜氏股由三个连续的离心（1201 g下5分钟），并在干净的FSW再悬浮彻底漂洗被馈送到幼虫之前，以除去藻类培养基这是富含必需金属的痕迹。
所有罐子在4天取样并11 D-受精后; 早期的计时组罐在25 D取样，并在13 D和27天postfertilisation晚定时基的罐子。期间的采样，在每个瓶中的水位降低到750 ml至增加幼虫密度并取每罐3×15毫升分装。罐子随机编号和所有测量进行盲关于治疗分类。使用解剖显微镜（放大40倍），每个等分试样进行测定的活幼虫数。每个活幼虫被分配到八个发展类（表3.2）之一。遇到40％的乙醇被保存了前十正常幼虫复合显微镜（放大倍数50倍）下拍照。当两个发育阶段被认为是正常的（例如，在25 D受精后两个6-武装plutei和8武装plutei被评为正常），只有属于最先进的发展阶段的幼虫（如8武装在25 D plutei）采取的形态测量。幼虫身体和臂长（平均R和L武器在可能的;表3.2）的测量，以及存在和后期幼虫阶段的雏形的大小时，使用软件ImageJ的完成。

結果 (中文) 3:

3.2.3幼虫的检测E. chloroticus幼虫饲养28 d在更新系统由2升的玻璃罐放在流动海水澡保持几乎恒定的温度。水的温度是16°C±1整个实验及pH值测定在每个罐子在一周和三周后受精。罐子轻轻的机动桨系统如斯特拉斯曼搅拌（1987），提供足够的氧。胚胎被放置在培养瓶内1小时后受精密度为1胚/毫升。所有玻璃器皿用热水洗净，浸泡至少4小时10%盐酸，清洗和浸泡在蒸馏水中12小时，在使用前冲洗搅拌摩擦焊。搅拌摩擦焊在坛子里恢复了每周三次的反虹吸。从受精后4 d，幼虫每换水与杜氏盐藻浓度为8000的培养细胞/毫升。盐股彻底冲洗三连续离心（1201 g，5 min）和resuspensions清洁FSW在喂养幼虫，去除藻类的培养基，在基本金属丰富的痕迹。所有的瓶子都在4 d和11 d后受精采样；在早期定时组陶罐25 d取样，在13 d和27 d postfertilisation在晚定时组罐。在取样过程中，每个罐的水位降到750毫升增加幼虫密度和3 x 15毫升等分试样每罐。瓶被随机编号，所有的测量都做了盲的治疗分类。在每一部分用解剖显微镜测定活幼虫数（40x放大）。每个活幼虫被分配到一个八个发展类（表3.2）。第一十正常幼虫遇到被保存在40%乙醇被拍到复合显微镜下（50x放大）。当两个发展阶段被认为是正常的（例如在25 d后施肥都6-armed plutei和8-armed plutei被评为正常），只有幼虫属于最先进的发展阶段（如8-armed plutei 25 d）进行形态测量。幼虫的身体和手臂的长度测量（R和L的手臂在可能的地方；平均表3.2）以及后期幼虫阶段的存在和大小的雏形进行软件ImageJ。