Name: resurrection of dormant daphnia magna: protocol and applications
Uploaded: 2018-01-19T13:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Resurrection of Dormant Daphnia magna: Protocol and Applications at JoVE.com

这项工作得到了 NERC 亮点赠款 (NE/N016777/1) 的支持。对虾有限公司, 环境科学服务, 环境变化研究中心, 伦敦大学学院取样和日期的沉积核心。

下面的 SOP 提供了一步一步的描述, 该协议用于复活大型蚤休眠卵, 包括取样的详细描述, 从沉积物中分离 ephippia, 以及建立克隆文化 (图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56637/56637fig1.jpg"> 图 1: 大型蚤的复活的分步指南. 天然淡水生境 (a) 中的沉淀物是用活塞取样 (B) 取样的。沉积核心 (C) 在1或 0.5 cm (D) 的增量层中切片。每层沉积物在黑暗和冷的情况 (4 ° c) 被存放在一个样品 zip 锁袋子 (E)。每层沉积物被称和被筛用地质筛子 (1 毫米和125µm 网格大小, F)。白色背景托盘是用来隔离蚤的麦格纳ephippia (G)。Decapsulated 休眠卵 (H) 被转移到培养皿中, 暴露在光照和温度刺激下诱导孵化。幼龟被转移到单独的罐子 (I) 以建立 isoclonal 线。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
1. 沉积物芯的取样
<ol><li>用活塞取样从湖泊或池塘中抽取沉淀物。本协议使用大本机27, 一个长度约为1.5 米的核心管, 内径为14厘米. 大本钟由一根绳子上的活塞和一个取样的头组成, 连杆被连接到其中, 把管子推进到沉淀物中。一个核心的捕手帮助支持的核心管时, 充满了泥沙。为了挤出沉积物, 一个框架保持核心管直立和固定, 和一个修改的瓶子千斤顶是用来推动活塞向上挤压过程中 (补充视频 1)。<ol>
<li>对于深度小于1米的浅水池塘, 使用一个不超过6厘米直径的有机玻璃重力取样, 将其手动推入沉积物中。</li>
		<li>对于深湖 (> 6 米的深度), 使用利文斯顿活塞取样28或单驱动器格里菲斯沉积取样与援助的一个抛锚的浮桥船。利文斯通式驱动杆活塞取样可用于30米深的水中, 以收集连续的一米的软到固结湖泊沉积物的驱动器。单一驱动器格里菲斯取样由一个简单而坚固的核心头, 连接标准的聚碳酸酯管到利文斯通驱动棒。取样被推挤入沉淀物与棒, 并且活塞提供必要的吸力为恢复沉积 (图 2)。</li>
		<li>为了检索连续的, 不受干扰的核心, 使用 vibracoring。这些取样在各种水深的基础上工作, 可以根据沉积物的岩性来检索不同长度的岩心样品。低振幅振动, 从 vibracore 头向下通过连接的桶或核心管液化沉积, 使核心桶连接到 vibracore 单位渗透到液化沉淀物。一些 vibracorers 是小型的, 轻量级的, 便携的, 其他是大型的重型单位, 只能从大型船舶部署。取样的选择取决于沉积物的岩性。</li>
	</ol>
</li>
	<li>使用平坦的金属表面 (辅助视频 1) 在1厘米或更小的增量层中水平地切片核心。像这里使用的沉积物取样, 旨在减少挤压时的静水压压力, 减少沉积层的扰动。当使用其他取样时, 每个沉积层的外皮都可以用切饼刀类的刀片来去除, 以限制层间的污染。</li>
	<li>收集每个沉积层在一个单独的取样袋 (补充视频 1), 并存储在黑暗和寒冷 (4 ° c) 的条件。</li>
	<li>从各个层收集至少5克的沉积物以进行辐射测年。由于辐射测年是对约会分析的详细描述的既定协议, 请参阅现有出版物12,13。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56637/56637fig2.jpg"> 图 2: 活塞取心程序的卡通.活塞取样, 中空管, 内部滑动密封 (活塞), 产生弱真空。当活塞接触到泥沙-水界面时, 重物将核心桶推入沉积物中, 真空使沉积物成核进入和移动到管道中, 而不干扰沉积层。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 沉积层的筛分
<ol><li>使用精密刻度, 权衡每一个沉积层, 以备将来参考。利用表面积和重量来计算湖中的物种密度。</li>
	<li>用两个地质筛子互相堆叠在一起, 筛出每个沉积层。第一个筛子有网格大小1毫米并且分离黏土、大无脊椎动物和微粒物质,例如种子、植物和昆虫, 从剩余的沉积。第二个筛子有一个网格125µm 并且分离D. manga ephippia 和小微粒从沉积的剩下的人 (补充录影 2)。</li>
	<li>
把收集到125µm 网筛上的泥沙分数的小等分转移到白色背景托盘上。根据沉积物的类型, 每一次可以转移较小或更大的沉积物等分。
	<ol>
<li>将小体积 (高达200毫升) 的培养基添加到取样白托盘中, 重新悬挂转移的沉积物分数, 并促进 ephippia 的眼部斑点 (图 3)。用于重沉积物的介质可以是 dechlorinated 自来水、钻孔水、组合29或 ADaM 介质 (Aachener Daphnien)30。此后, "媒介" 一词将被用来指任何或所有列出的解决方案。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56637/56637fig3.jpg"> 图 3: ephippium蚤休眠在开封后立即进行蚤类的孵化。ephippium (A)、内卵膜 (B) 和休眠卵 (C) 显示为.缩放条 = 500 µm。
3. Ephippia 和孵化的开封
<ol><li>用一次性的巴斯德吸管或使用显微解剖钳, 将单个的 ephippia 转移到培养皿中, 填充10毫升培养基。每层沉积物至少使用一个培养皿。</li>
	<li>在一个立体声显微镜下, decapsulate 每 ephippia 使用显微解剖钳, 强迫打开甲壳质的情况 (补充视频 3)。小心地取出休眠的鸡蛋内膜, 注意不要扰乱鸡蛋, 然后把它们转移到用巴斯德吸管填充培养基的培养皿中。开封增加了D. 麦格纳中的孵化成功率; 但是, 可能不需要, 也可能对其他蚤类物种产生较小的 ephippia 的挑战。</li>
	<li>将 decapsulated 的卵暴露于全谱长日照光周期 (16:8 光: 深色) 和高温 (20 ±1° c), 以诱导在受控温度设备 (孵化器) 或房间内孵化。孵化发生在48小时和几个星期之间 (最多四;辅助视频 4)。在没有开封的情况下, 直接暴露 ephippia 到孵化刺激 (长日光周期光照 (16:8 光: 暗) 和高温 (20 ±1° c))。</li>
</ol>4. 建立 Isoclonal 的蚤系列
<ol><li>通过将单个的D. 麦格纳从步骤3.3 转移到用一次性的巴斯德吸管填充200毫升培养基的分离罐中, 建立单幼龟的 isoclonal 线。每个个体在基因上是不同的, 是性重组的结果。</li>
	<li>在10±1° c, 16:8 光: 黑暗政权, 美联储周刊与0.2 毫克 C/L 的小球藻或其他绿藻 (例如,栅菌) 的库存条件下, 无限期地维护 isoclonal 线。每三周更新一次培养基。储存条件可能随温度、摄食制度和物种而变化。</li>
</ol>5. 重点研究
注: 两项关键研究的描述, 其中有复活的D. 麦格纳(子) 从湖环沉积档案馆 (丹麦) 的人口被用来评估气候变暖的进化反应。三 (分) 种群从以下时间段中复活: 1960-1970、1970-1985 和 > 1999。D. 麦格纳从沉积存档中成功孵化的范围介于11和58% 之间 (图 4)。从每个 timeperiod 获得的幼龟, 选择了一个随机子集, 用于这里描述的两个关键研究。这些研究的目的是评估 (分) 从不同时间段复活的人群在温度梯度上是否表现出与健康相关的生活史特征的差异 (5.1), 以及他们是否有不同的竞争能力 (5.2)在接触到变暖之后
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56637/56637fig4.jpg"> 图 4: 从湖环取样的沉积档案中孵化成功.重点研究了湖环沉积档案中成功幼龟的比例。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>
共同的庭院实验
	<ol>
<li>将十只幼龟每 (分) 种群从砧木养殖转移到普通花园条件:16 ° c;长周期 (16:8 光: 黑暗的政权);每日以0.8 毫克的小球藻为食, 每第二天更新培养基。在普通花园条件下保持幼龟至少两代 (ca 45 天)。常见的花园环境减少了母体效应的干扰, 并使幼龟的繁殖同步。</li>
		<li>在第二个巢的释放到巢室, 转移成年女性从第二代到500毫升罐子充满媒介直到他们释放第二个巢幼仔。</li>
		<li>随机转移 24–48 h 的个体幼仔, 出生于第二代第二巢, 到100毫升罐子充满介质, 并暴露于以下实验条件:18 ° c (当前温度在湖) 和24° c (变暖, 温度预测由气专委26为即将到来的100年), 16:8 光: 黑暗政权, 和饲料每日0.8 毫克 C/升的Chlorellavulgaris。</li>
		<li>在每个实验动物上, 以显微镜作为头部与尾部底部之间的距离 (图 5) 来测量成熟时的大小。拍摄每只动物, 然后用一个合适的图像软件分析它的大小。</li>
		<li>测量成熟时的年龄: 在孵化室中第一次观察卵子的日子。</li>
		<li>测量死亡率: 在实验中灭绝的个体的数量。</li>
		<li>测量繁殖力: 第一和第二克隆繁殖后代的总数量。</li>
		<li>用欧拉方程估计人口增长率 (1): <img alt="Equation" src="/files/ftp_upload/56637/56637eq1.jpg">(1) 其中, lx 是生存期的比例x, bx 是每个存活个体在年龄段x中产生的新生儿数, r 是自然增量的固有速率。</li>
		<li>使用商业可用的软件进行统计分析。在这里, 使用 R31来绘制每个生命历史特征的反应规范 (在不同环境中的单基因型表达式), 使用 ggplot2 包。通过生存分析计算每个人口的死亡率 (R 包 rms; https://cran.r-project.org/web/packages/rms/rms.pdf)。最后, 对 R31中的方差 (方差分析,表 2) 进行评估, 以确定温度对性状的影响是否可以通过进化 (种群之间的差异)、可塑性 (对治疗的反应) 或进化来解释可塑性 (人口 x 治疗)。</li>
	</ol>
</li>
	<li>
竞赛实验
	<ol>
<li>将每人口十只幼龟从砧木养殖转移到常见的花园条件:20 ° c, 长光周期 (16:8 光照: 暗区), 每天以0.8 毫克的小球藻为食,并每第二天更新培养基。保持至少两代 (ca 45 天) 的共同菜园条件, 以减少对母体影响的干扰。</li>
		<li>随机分配五青少年24至 48 h 从第二个巢每孵化到实验 mesocosms (20 L 塑料水族馆充满中等), 在 triplicates, 在密度10动物/升。</li>
		<li>将 mesocosms 暴露于24° c、16:8 L: D 机制, 并每日以0.8 毫克碳/l 的Chlorellavulgaris在受控温度室或孵化器中, 至少四周 (> 3 克隆世代)。</li>
		<li>剔除每个围每周, 刷新10% 的媒体, 以模拟人口动态,蚤可能会遇到的自然环境。通过从每个水族箱中取出已知的培养基和动物 (在这种情况下为1.2 升), 并用新鲜培养基取代被扑杀的体积来进行扑杀。在实验动物达到成熟期后开始扑杀制度 (10 天)。</li>
		<li>在第四周结束时, 从每个围取样32动物来评估基因型成分的变化, 与最初的接种。<ol>
<li>在离心管中放置单独的水蚤, 并在巴斯德吸管的帮助下去除多余的液体。</li>
			<li>在-80 ° c 的温度下, 将单个管子冷冻在液氮中。</li>
			<li>利用现有的协议和制造商的说明, 从单个个体提取基因组 DNA。</li>
			<li>使用大量的遗传标记来放大提取的 DNA, 足以提供一个独特的位基因型每孵化。在这里, 8 微卫星在一个单一的复用 (M01,表 1) 被使用了在建立的协议以后32,33。</li>
			<li>片段分析仪上的基因型放大片段。</li>
			<li>使用合适的尺寸标准对商业或自由可用的软件进行碎片分析。</li>
			<li>在实验结束时评估基因型成分, 将32个体与一组微卫星位点描述为33, 并在实验结束时与初始接种相比, 计算每种基因的频率。</li>
		</ol>
</li>
	</ol>
</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56637/56637fig5.jpg"> 图 5: 成年女性大蚤. 成年雌性的大蚤, 在巢室中有孤雌卵。头部和尾部的底部之间的距离用来测量动物的大小。红线表示尺寸测量。缩放条 = 500 µm。

作者没有透露

由于水的高导热性, 在面对全球变暖的情况下, 淡水生态系统的生物多样性损失的风险比地球生态系统高34。因此, 重要的是要了解这些生态系统中的梯形物种的反应, 并确定应对机制, 以生存热应力。在物种和群落层面上对这些机制的理解可以帮助预测物种如何受到全球变暖的影响, 以及对个体物种的作用如何与其他营养水平有关。最终, 了解全球变暖反应机制有助于确定补救战略, 以减少灭绝。
这里的案例研究表明, D. 麦格纳对温度升高的反应是由生命历史特征中的可塑性普遍介导的, 而仅对温度增加的反应并不会造成明显的健身成本, 至少在这里研究的人口。生命史特征的高可塑性是由在变暖的情况下 (子) 种群的竞争能力的不显著差异所支撑的。然而, 对多个种群进行长期的竞争试验可能是推广这些发现的必要条件。
休眠阶段的复活提供了一个空前的资源来研究一个物种的进化机制, 通过时间10。浮游动物物种受益于快速世代 (约2周), 和休眠阶段的生存能力, 允许祖先竞争 headtohead 自己的后代, 或 ' 重放 ' 的进化, 从不同的过去的状态开始。复活生态学基本上能够调查某一特定的进化结果是否取决于某个先前的事件。目前, 在实验室实验中, 利用微生物对其进行了鉴定, 并将其与进化后代6进行比较分析, 并对其进行了 "祖先系" 的冷冻和复苏。然而, 与实验室有机体一起工作的一个主要限制是, "祖先状态" 是已经转移的基线。休眠阶段的研究允许取样从时间早任何应力事件 (例如, 原始环境条件) 和测量进化轨迹从不受干扰的环境条件到不同的过去状态直到现代。近年来, 对复活或静止的浮游动物阶段 DNA 多态性的研究为过去的人口统计学和自适应过程提供了重要的洞察力, 这有助于当代人口的遗传构成14,16,25,33,35,36. 随着高吞吐量测序技术的更高的可及性, 可以对复活或静止阶段的基因组和转录序列进行排序, 并在进化的种群中积累的遗传变化的类型和数量时间测量。
本文提出的复活 SOP 在两个层次的多学领域有着重要的应用。多学技术可以应用于复活标本, 允许对参与环境选择压力的自适应反应的分子元素进行详尽的分析。此外, 学技术可应用于 decapsulated, 但仍处于休眠阶段。到目前为止, 高通量测序技术在休眠阶段的应用受到大量输入材料的限制。这些限制正在被解除37。随着输入材料的降低和学的进展, 整个基因组测序 (WGS) 现在可以从 1 ng 或几个起始材料的 pg38。使用全基因组扩增 (WGA) 和全转录扩增技术, 使 DNA 和 RNA 从非常少量的组织中富集, 已经彻底改变了基因39,40和医学研究41。这些技术适用于 decapsulated 休眠卵, 使超过休眠阶段的生存能力和调查延长的时间段 (例如, 世纪) 的限制。
产生休眠阶段的无脊椎动物群落的复活使群落的历史与自然景观的已知变化相一致, 或通过对沉积物 orsoils2的分析推断出环境变化。对环境变化作出反应的社区变化的分析为我们提供了量化生态进化反馈的能力42 , 它对人口持久性有重大影响43, 营养交互作用44, 社区程序集45, 以及生态系统功能和服务的更改46。最后, 对环境变化的生物反应的准确预测对于指导生物多样性的保护是至关重要的47。目前的预测模型在这方面不准确, 因为它们没有考虑到重要的生物机制, 如人口统计学、散布、进化和物种相互作用。了解这些过程是如何随着时间的推移而变化的, 并将这些信息作为预测模型的前期使用, 将提高我们在环境变化2面前预测物种和社区持久性的能力。
在这里提出的 SOP 的应用并非没有挑战。复活休眠阶段的主要限制是需要专门的设备进行取样。此外, 从沉积物筛选到克隆文化的建立, 整个过程需要大量的时间。
这里介绍的一些 SOP 步骤很容易转移到其他蚤类物种。这些是: 取样, 建立克隆线, 和实验设计。然而, SOP 的其他步骤可能需要进一步优化为研究中的物种量身定做。开封通常应用于D. 麦格纳标本, 以提高孵化成功率。然而, 这种方法可能不适合较小的标本。孵化刺激也可能在物种之间变化48和同种标本49。因此, 在应用于其他甲壳类之前, 可能需要对 SOP 的孵化步骤进行ad hoc优化。虽然D. 麦格纳种群的孵化成功来自湖环 (30.5% 横跨沉积档案), 但与以前的结果是一致的49, 但孵化成功率随沉积物的保存状态而变化, 物种50,51, 以及沉积物的地理来源48。未来的研究的机制, 调节进入和进展, 通过阶段的滞育是需要确定最佳孵化刺激适合不同物种。
最后, 研究系统的背景知识, 特别是有兴趣的物种的存在的时间, 是可取的。这可以通过历史记录来实现。如果历史记录不可用, 则在取样前对湖泊沉积物表层进行抽样和筛选是可取的, 尽管它可能只提供最新历史资料。

长期研究对于理解自然界的生态和进化过程以及评估物种在环境变化过程中的反应和坚持是至关重要的1。这是因为生态进化过程发生在几代人之间, 环境的变化发生在很长的时间跨度内。此外, 长期研究提供了关键的经验数据, 提高预测建模的准确性, 预测自然生态系统对环境变化的进化响应2。这些模型的准确性对于实施管理和保护战略以保护生物多样性和生态系统服务至关重要。
除了少数例外情况 (例如、加拉帕戈斯达尔文雀科3和藻类4) 外, 长期研究主要限于在实验室中可以传播的短时间内的物种, 它们可在5、6,7,8. 因此, 支撑进化动力学的过程仍然难以捉摸。由于与访问时态样本相关的逻辑困难, 经验数据在空间中比在时态环境中更频繁地进行研究, 而时间生态进化过程是从空间数据中推断或建模的。这种方法被称为 "时空替换"9, 藉以空间作为研究时间演化动力学的替代品。"时空替代" 的主要限制是, 不同空间尺度的适应率与同一种群的时间变化不同;因此, 基于空间替换时间的推论是有偏向的10。
一个强有力的替代方法, 允许研究自然生态系统中的进化动力学, 是对产生休眠阶段的物种的生态和遗传变化的分析11。这些休眠阶段积累形成分层的生物档案, 可以准确日期和 paleolimnologically 特征12,13。重要的是, 这些休眠阶段可以被复苏和用于实验室实验, 在那里他们对环境变化的进化反应可以直接测量。历史种群可以与他们的现代进化后代进行竞争, 以研究适应环境变化的变化和基因的功能演变14,15,16。
休眠阶段包括种子、囊肿、孢子和卵库。虽然第一项关于复苏休眠卵的研究可追溯到二十世纪八十年代后期的17, 而少数研究在二十世纪九十年代早期的18、19中应用了这项技术, 但复活生态学的领域已经正式由海伦·凯尔富特和联合工人的精纸建立 1999年20。这一做法主要适用于淡水物种的湖泊重建17,21,22。但是, 尚未提供 SOP。在这里, 一步一步描述的复活协议适用于休眠卵的浮游动物物种大型蚤提供, 从沉积物的取样到建立克隆文化从幼龟。本文讨论了易于转移到其他种类的蚤类的 SOP 步骤, 以及可能需要额外优化的步骤。
蚤型是淡水 zooplankters 目前在大多数激流生境23 。蚤类物种要么是强制无性繁殖, 要么是周期性 parthenogens。D. 麦格纳是一种周期性的 parthenogen, 它在有利的环境条件下复制无性24。当环境条件恶化时, 雄性的生产发生, 性重组导致受精卵的形成, 这种休眠状态被称为 ephippium 的甲壳素案例所保护。当有利的环境条件返回时, 这些休眠卵中的一部分会孵化。然而, 在休眠的卵子库中, 有很大一部分从来没有机会孵化, 从而随着时间的推移建立生物档案。休眠阶段仍然埋藏在湖泊和池塘的沉积物中, 可以在延长的时间内对进化动力学的研究复活。由于休眠卵的D. 麦格纳是性重组的结果, 他们是一个很好的代表性的自然遗传多样性的物种25。此外, 它们可以通过克隆繁殖在实验室保持。这些特性提供了基因模型生物体的独特优势, 同时保留了自然遗传多样性。
两个关键的研究, 以证明的优势, 直接比较历史和现代后裔的相同人口的D. 麦格纳体验环境选择压力随着时间的推移。D. 麦格纳标本是从湖环 (丹麦) 中复活的, 浅 (5 米深; 表面22公顷) 混合池, 在一段时间内, 平均温度和热浪发生了增加。D. 麦格纳(子) 种群是沿着这一历时60年的时间梯度 (1960–2005) 复活的, 并研究了对温度变暖的进化反应。在一个共同的庭院实验的第一项研究中, 根据未来100年政府间气候变化专门委员会的预测, 测量了与 +6 ° c 的气温升高有关的与健身相关的生活史特征的变化。26. 在第二项研究中, 围实验被用来测量三 (分) 种群在变暖下的竞争能力。这些实验表明, 在变暖作为唯一的压力下, 所有的生命史特征和种群表现出高度的可塑性, 具有同等的竞争能力。这些发现表明, 变暖作为一个单一的压力并不会造成很大的健身成本, 至少在这里的人口研究。

<table><tbody><tr><td>sampling bags</td><td>Fisher Scientific</td><td>11542783</td><td>Sampling bag revolve round wires closure system and safety tabs sterile polyethylene with writing area clear 89&amp;micro;m thickness 140mm x 229mm, 720mL Fisherbrand</td></tr><tr><td>piston corer</td><td>ENSIS ltd</td><td>na</td><td>Long, heavy tube plunged into the sea, lake, pond floor to extract samples of mud sediment. Piston corers have a viariable diameter and are generally in PVC</td></tr><tr><td>precision scale</td><td>Veritas-M124A</td><td>TLP-50</td><td>Analytical Balance</td></tr><tr><td>geological sieve</td><td>UKGE limited</td><td>SV7521</td><td>200 mm diameter geological sieve - 1 mm mesh</td></tr><tr><td>geological sieve</td><td>UKGE limited</td><td>SV7525</td><td>200 mm diameter geological sieve - 0.125 mm mesh</td></tr><tr><td>white sampling tray</td><td>nhbs</td><td>http://www.nhbs.com/title/view/159614?ad_id=1509</td><td>Standard mulipurpose lab trays</td></tr><tr><td>pasteur pipette</td><td>Globe Scientific inc.</td><td>138020B</td><td>Transfer Pipet, 1.7mL, General Purpose, 87mm, Bulb Draw - 0.9mL</td></tr><tr><td>stereo microscope</td><td>nikon</td><td>smz800</td><td>Microscope with magnification range 1x -8x linked to a camera control unit</td></tr><tr><td>petri dish</td><td>EduLab</td><td>153-533</td><td>Sterile 90mm diameter plastic petri dish</td></tr><tr><td>glass jars</td><td>compak</td><td>Round Jam Jars 4oz</td><td>100 mL jars</td></tr><tr><td>glass jars</td><td>compak</td><td>Atum Jars/ Bonta Jar 10oz</td><td>200 mL jars</td></tr><tr><td>glass jars</td><td>bottlecompanysouth</td><td>500ml Food Jam Jar With Twist Off Lid</td><td>500 mL jars</td></tr><tr><td>statistical software R</td><td>https://cran.r-project.org/</td><td>na</td><td>Free online GNU&amp;nbsp; language and environment for statistical computing and graphics</td></tr><tr><td>microdissection forceps</td><td>Fisher Scientific</td><td>41122405</td><td>Fine point stainless steel forceps for microdissections</td></tr><tr><td>image software</td><td>https://imagej.nih.gov/ij/index.html</td><td>na</td><td>Open source ImageJ image processing toolkit written in Java</td></tr><tr><td>mesocosm</td><td>amazon</td><td>na</td><td>Nobby Fauna-Box III, 41 x 23 x 29 cm, 20.0 Liter</td></tr><tr><td>mirocentrifuge tubes</td><td>Sigma_Aldrick - Merck</td><td>Z606340</td><td>premium microcentrifuge tubes 1.5 mL</td></tr><tr><td>AGENCOURT DNAdvance</td><td>Beckman Coulter</td><td>A48705</td><td>DNA extraction kit</td></tr><tr><td>size standard</td><td>Thermo Fisher Scientific</td><td>4322682</td><td>LIZ500 - Size standard compatible with ABI sequencers</td></tr><tr><td>ABI3032 sequencer</td><td>ABI</td><td>na</td><td>Sequencer used to perform fragment analysis or sanger sequencing</td></tr></tbody></table>

resurrection of dormant daphnia magna: protocol and applications

长期研究能够识别出在较长时期内发生的生态进化过程。此外, 它们还提供了重要的经验数据, 可用于预测模型预测自然生态系统对未来环境变化的进化反应。然而, 除了少数例外情况, 长期研究是稀缺的, 因为与访问时态样本相关的后勤困难。在实验室或受控围实验中经常研究时间动力学, 并通过特殊的研究来重建野生种群的进化。
在这里, 提供了一个标准操作程序 (SOP) 来恢复或复活休眠的水蚤, 一个广泛的浮游动物的重点物种在水生生态系统, 以戏剧性地推进国家的艺术纵向数据收集自然系统。在 1999年, 海伦·凯尔富特和联合工作者定义了复活生态学的领域, 尽管第一次尝试孵化滞浮游动物的卵可追溯到二十世纪八十年代代末。自从海伦·凯尔富特的开创性论文, 复活浮游动物的方法越来越频繁地被应用, 虽然在实验室之间传播仅通过直接知识转移。这里描述了一个 SOP, 它提供了一个逐步的协议, 关于复活休眠的大型蚤卵的做法。
两项关键研究提供的是, 对复活的大蚤种群对变暖的适应性反应进行了测量, 利用了在相同环境中研究历史和现代种群的能力。最后讨论了下一代测序技术在恢复或静止阶段的应用。这些技术提供了前所未有的权力, 解剖的过程和机制的演变, 如果适用于人口已经经历了变化的选择压力随着时间的推移。

长期的经验数据对于理解进化动力学和自然种群的持续性是至关重要的。这类数据通常具有挑战性, 因为与访问时态样本和要求长期进行数据收集有关的后勤困难。在这里介绍的两项主要研究中, 在进化时期提供了对淡水生态系统中中央 zooplankter 温度反应的经验证据。这是通过使用分层休眠蛋库提供了机会, 研究历史人口和他们的现代后裔对环境压力在共同实验设置的反应。
普通园林实验 普通菜园实验表明, 所有生命历史特征都响应了温度 (图 6和 图 7)。方差分析显示, 所有 (子) 种群都通过可塑性 (表 2) 对温度做出反应, 但死亡率没有反应。进化变化的证据 ((分) 种群之间的差异) 只在人口增长率 (表 2) 中观察到, 在24° c (图 6) 的三 (子) 人口中的两个人中, 显著增加。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56637/56637fig6.jpg"> 图 6: 普通菜园试验.与普通菜园和当前温度 (18 ° c) 相比, 在温度变暖 (24 ° c) 下, 对每个 (子) 种群的生命史特征 (生育力、大小和年龄) 和人口增长率 (r) 的反应准则都显示出来。使用欧拉方程 (1) 计算了人口增长率 "r"。显示置信区间。(子) 填充颜色编码: (i) 蓝色: 1960-1970;(二) 绿色: 1970–1985;(iii) 红色: > 1999。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56637/56637fig7.jpg"> 图 7: 死亡率.与现代温度制度 (18 ° c) 相比, 每 (次) 人口死亡率 (1960-1970; 1970-1985; > 1999) 在升温 (24 ° c) 下显示。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
围实验 经过四周的选择, 以24° c 的升温为代表, 三 (次) 种群的频率没有发生显著变化 (χ2 = 0.55, P = 0.76), 与初始接种 (图 8)。在围试验中接种的30种基因型中, 大多数是在选择四周后 (图 9) 中确定的。具体来说, 70% 的接种基因型被恢复, 符合泊期望恢复至少一个代表的每一个基因型在样本的32人。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56637/56637fig8.jpg"> 图 8: 竞争实验-人口频率.四 (25th和 75th) 的总体平均值为D. 麦格纳的三 (分) 填充, 在围竞争实验 (24 ° c) 的四周后, 与初始等频率 (在开始)。(Sub) 填充颜色编码, 如图 6所示。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56637/56637fig9.jpg"> 图 9: 竞争实验-基因型频率.基因型频率-平均中位数和四 (25th和 75th), 显示后四周的接触升温 (24 ° c) 与初始等频率的基因型 (虚线)。x 轴上的名称是被接种的基因型 ID, 按 (分) 种群 (蓝色, 1960-1970; 绿色, 1970-1985; 红色, > 1999) 分组。<a href="//cloudflare.jove.com/files/ftp_upload/56637/56637fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>轨迹</td>
			<td>一个</td>
			<td>尺寸范围 (bp)</td>
			<td colspan="2">底漆 (5 "-3")</td>
			<td>染料标签</td>
			<td>重复主题</td>
			<td>tm</td>
		</tr>
<tr>
<td rowspan="2">B008</td>
			<td rowspan="2">HQ234154</td>
			<td rowspan="2">150–170</td>
			<td colspan="2">F: TGGGATCACAACGTTACACAA</td>
			<td rowspan="2">死者</td>
			<td rowspan="2">tc)9
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: GCTGCTCGAGTCCTGAAATC</td>
		</tr>
<tr>
<td rowspan="2">B030</td>
			<td rowspan="2">HQ234160</td>
			<td rowspan="2">154–172</td>
			<td colspan="2">F: CCAGCACACAAAGACGAA</td>
			<td rowspan="2">宠物</td>
			<td rowspan="2">遗传)11
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: ACCATTTCTCTCCCCCAACT</td>
		</tr>
<tr>
<td rowspan="2">B045</td>
			<td rowspan="2">HQ234168</td>
			<td rowspan="2">118–126</td>
			<td colspan="2">F: GCTCATCATCCCTCTGCTTC</td>
			<td rowspan="2">斯内德</td>
			<td rowspan="2">tg)8
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: ATAGTTTCAGCAACGCGTCA</td>
		</tr>
<tr>
<td rowspan="2">B050</td>
			<td rowspan="2">HQ234170</td>
			<td rowspan="2">234–248</td>
			<td colspan="2">F: TTTCAAAAATCGCTCCCATC</td>
			<td rowspan="2">6FAM</td>
			<td rowspan="2">gaa)6
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: TATGGCGTGGAATGTTTCAG</td>
		</tr>
<tr>
<td rowspan="2">B064</td>
			<td rowspan="2">HQ234172</td>
			<td rowspan="2">135–151</td>
			<td colspan="2">F: CTCCTTAGCAACCGAATCCA</td>
			<td rowspan="2">6FAM</td>
			<td rowspan="2">tc)8
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: CAAACGCGTTCGATTAAAGA</td>
		</tr>
<tr>
<td rowspan="2">B074</td>
			<td rowspan="2">HQ234174</td>
			<td rowspan="2">196–204</td>
			<td colspan="2">F: TCTTTCAGCGCACAATGAAT</td>
			<td rowspan="2">斯内德</td>
			<td rowspan="2">gt)9
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: TGTGTTCCTTGTCAACTGTCG</td>
		</tr>
<tr>
<td rowspan="2">B096</td>
			<td rowspan="2">HQ234181</td>
			<td rowspan="2">234–240</td>
			<td colspan="2">F: GGATCTGGCAGGAAGTGGTA</td>
			<td rowspan="2">死者</td>
			<td rowspan="2">交流)15
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: TTGAACCACGTCGAGGATTT</td>
		</tr>
<tr>
<td rowspan="2">B107</td>
			<td rowspan="2">HQ234184</td>
			<td rowspan="2">250–274</td>
			<td colspan="2">F: GGGGTGAAGCATCAAAGAAA</td>
			<td rowspan="2">宠物</td>
			<td rowspan="2">ct)8
</td>
			<td rowspan="2">56</td>
		</tr>
<tr>
<td colspan="2">R: TGTGACCAGGATAAGAGAAGAGG</td>
		</tr>
</tbody></table>表 1: 微卫星复用.NCBI 加入号 (a)、复用信息、pcr 引物序列、pcr 大小范围、重复母题、用于标记前引物的染料以及退火温度 (Tm)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">Pop 增长率 (r)</td>
			<td>df</td>
			<td>f</td>
			<td>p</td>
		</tr>
<tr>
<td colspan="2">演变 (流行音乐)</td>
			<td>2</td>
			<td>30.309</td>
			<td>< 0.001</td>
		</tr>
<tr>
<td colspan="2">塑性 (温度)</td>
			<td>1</td>
			<td>531.546</td>
			<td>< 0.001</td>
		</tr>
<tr>
<td colspan="2">演化.可塑性 (流行 x 温度)</td>
			<td>2</td>
			<td>65.137</td>
			<td>< 0.001</td>
		</tr>
<tr>
<td colspan="2">死亡率</td>
			<td>df</td>
			<td>f</td>
			<td>p</td>
		</tr>
<tr>
<td colspan="2">演变 (流行音乐)</td>
			<td>2</td>
			<td>2.234</td>
			<td>0.1162</td>
		</tr>
<tr>
<td colspan="2">塑性 (温度)</td>
			<td>1</td>
			<td>2.679</td>
			<td>0.1071</td>
		</tr>
<tr>
<td colspan="2">演化.可塑性 (流行 x 温度)</td>
			<td>2</td>
			<td>1.8657</td>
			<td>0.164</td>
		</tr>
<tr>
<td colspan="2">繁殖</td>
			<td>df</td>
			<td>f</td>
			<td>p</td>
		</tr>
<tr>
<td colspan="2">演变 (流行音乐)</td>
			<td>2</td>
			<td>1.8852</td>
			<td>0.1633</td>
		</tr>
<tr>
<td colspan="2">塑性 (温度)</td>
			<td>1</td>
			<td>6.8934</td>
			<td>0.0117</td>
		</tr>
<tr>
<td colspan="2">演化.可塑性 (流行 x 温度)</td>
			<td>2</td>
			<td>1.6511</td>
			<td>0.203</td>
		</tr>
<tr>
<td colspan="2">到期时的大小</td>
			<td>df</td>
			<td>f</td>
			<td>p</td>
		</tr>
<tr>
<td colspan="2">演变 (流行音乐)</td>
			<td>2</td>
			<td>0.211</td>
			<td>0.8106</td>
		</tr>
<tr>
<td colspan="2">塑性 (温度)</td>
			<td>1</td>
			<td>11.1361</td>
			<td>0.0017</td>
		</tr>
<tr>
<td colspan="2">演化.可塑性 (流行 x 温度)</td>
			<td>2</td>
			<td>0.6586</td>
			<td>0.5225</td>
		</tr>
<tr>
<td colspan="2">成熟年龄</td>
			<td>df</td>
			<td>f</td>
			<td>p</td>
		</tr>
<tr>
<td colspan="2">演变 (流行音乐)</td>
			<td>2</td>
			<td>0.7811</td>
			<td>0.4637</td>
		</tr>
<tr>
<td colspan="2">塑性 (温度)</td>
			<td>1</td>
			<td>8.0764</td>
			<td>0.0066</td>
		</tr>
<tr>
<td colspan="2">演化.可塑性 (流行 x 温度)</td>
			<td>2</td>
			<td>0.088</td>
			<td>0.9159</td>
		</tr>
</tbody></table>表 2: 方差分析(方差分析).方差检验的分析是否生命史特征的变化和人口增长率的复活 (子) 人口暴露于变暖是解释的进化适应 (人口), 可塑性 (温度处理), 及其相互作用期限 (塑性的演变)。重要的p值 (p< 0.05) 以粗体显示。
补充视频 1: 沉积物核心取样一个大本取样的使用显示。大本钟是一个核心管约1.5 米的长度与内部管直径14厘米。它包括一个活塞在绳索和取样头, 标尺附有驾驶管子入沉积。核心捕手是用来支持从一个小容器部署的核心管。活塞通过重力压力向下推入沉积物。一个框架是用来支持的核心管在挤压过程中进行了使用修改瓶千斤顶, 推动活塞向上。每个沉积层都在一个平坦的金属表面收集, 并转移到透明的取样袋长期储存 [黑暗和寒冷 (4 ° c) 的条件]。<a href="http://cloudflare.jove.com/files/ftp_upload/56637/Supplementary_video1_Sampling.mp4">请单击此处下载此文件.</a>
补充视频 2: 沉淀物筛分.筛分泥沙所需的设备是精密刻度、白色取样塔和地质筛。从每个沉积层, 至少 5 g 保留为辐射约会。剩余的沉积物被用来隔离 ephippia。沉积物通过二个地质筛子被筛, 一个与1毫米和一秒钟以125µm 网格大小, 堆积在彼此之上。将培养基倒入 1 mm 网筛上, 以分离粘土、大无脊椎动物和颗粒物。介质浇在第二筛子上, 用125µm 网格将D. 麦格纳ephippia 和小颗粒物分开。等分沉积物然后转移到一个白色取样盘。D. 麦格纳ephippia 在白色背景托盘中被眼睛发现。每层的 Ephippia 都是在不同的培养皿中收集的。<a href="http://cloudflare.jove.com/files/ftp_upload/56637/Supplementary_video2_Sieving_sediment.mp4">请单击此处下载此文件.</a>
辅助视频 3: 开封.在显微镜下, D. 麦格纳ephippia 用显微解剖钳打开, 对甲壳质壳的脊柱施加压力。内卵膜是微妙地去除和休息鸡蛋轻轻转移与巴斯德吸管到一个培养皿含有10毫升的培养基。<a href="http://cloudflare.jove.com/files/ftp_upload/56637/Supplementary_video3_Decapsulation.mp4">请单击此处下载此文件.</a>
辅助视频 4: 剖面线在暴露于长周期和20° c 后, 胚胎发育在48小时到几周之间恢复。发育完成后, 胚胎从蛋壳中挣脱出来, 在培养基中自由游动。<a href="http://cloudflare.jove.com/files/ftp_upload/56637/Supplementary_video4_Hatching.mp4">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Resurrection of Dormant Daphnia magna: Protocol and Applications at JoVE.com

Resurrection of Dormant Daphnia magna: Protocol and Applications

长期研究对于理解进化过程和适应机制至关重要。一般来说, 这些研究需要在研究人员生命期之外做出承诺。在这里, 一个强大的方法被描述, 极大地推进了国家的艺术数据收集, 以产生纵向数据的自然系统。

休眠蚤的复活: 协议和应用

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

resurrection-of-dormant-daphnia-magna-protocol-and-applications

Research

JoVE Journal

Environment

Resurrection of Dormant Daphnia magna: Protocol and Applications

18.2K 次观看.  University of Birmingham. 长期研究对于理解进化过程和适应机制至关重要。一般来说, 这些研究需要在研究人员生命期之外做出承诺。在这里, 一个强大的方法被描述, 极大地推进了国家的艺术数据收集, 以产生纵向数据的自然系统。

视频: Resurrection of Dormant Daphnia magna: Protocol and Applications

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.

This article describes a simple and reproducible protocol to manipulate dissolved oxygen conditions in a laboratory setting for animal behavior studies. This protocol may be used in both teaching and research laboratory settings to evaluate organismal response of macroinvertebrates, fishes, or amphibians to changes in dissolved oxygen concentration.

一个简单的方法来操作的动物行为观察溶解氧

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal ...

科研

环境科学

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

鱼类生殖生态学研究的基本方法

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

行为学

用于果 蝇 菌株长期保存的 PGC 冷冻保存

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15&#160;agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 &#177; 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 &#177; 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

作者聚焦：PGC 冷冻保存 – 一种长期保存果蝇菌株的替代方法 

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

果蝇菌株的原始生殖细胞冷冻保存和复兴

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

遗传学

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

生物学

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

培养的 果蝇 脑外植体中的神经干细胞再激活

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

休眠蚤的复活: 协议和应用

摘要

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此视频中的章节

一个简单的方法来操作的动物行为观察溶解氧

藤壶Balanus improvisus作为海洋模型的培养和基因表达

鱼类生殖生态学研究的基本方法

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

在完整的果蝇幼虫体内的亚细胞分辨率成像

药理和功能的遗传分析处理涡虫的再生日本三角涡虫

的透的一种方法

培养的果蝇脑外植体中的神经干细胞再激活

休眠蚤的复活: 协议和应用

摘要

探索更多视频

此视频中的章节

一个简单的方法来操作的动物行为观察溶解氧

藤壶Balanus improvisus作为海洋模型的培养和基因表达

鱼类生殖生态学研究的基本方法

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

在完整的果蝇幼虫体内的亚细胞分辨率成像

药理和功能的遗传分析处理涡虫的再生日本三角涡虫

的透的一种方法

培养的 果蝇 脑外植体中的神经干细胞再激活

培养的果蝇脑外植体中的神经干细胞再激活