特别感谢以前的嗜 霉 菌畜群饲养员（Jacob E. Bliss、Paula Bonazinga、Anne W. Kerrebrock、Ingrid M. Mercer、Heidi S. Smith）和研究人员（特别是 Robert Baird、Michael S. Foulk、Donna Kubai、John M. Urban、Yutaka Yamamoto）对饲养方案进行了微调。Helen V. Crouse、Natalia Gabrusewycz-Garcia、Reba M. Goodman、Charles W. Metz 和 Ellen Rasch 提供了关于 嗜粪芽孢杆菌 护理的初步说明。感谢 Yukiko Yamashita 和 Anne W. Kerrebrock 接管 Bradysia （Sciara） 库存中心。非常感谢以下人员对图表的帮助准备:Brian Wiegmann（图 1）、John M. Urban（图 4 上面板）、Laura Ross（图 4 下面板）、Yutaka Yamamoto（图 5 左面板）、Leo Kadota（图 7 和 图 8）。非常感谢 Ava Filiss 和布朗大学多学科实验室在摄影和拍摄方面的帮助。感谢 Robert Baird 对手稿的评论。我们对 嗜粪芽孢杆菌 的研究和维护得到了 NIH 和 NSF 的支持，包括 NIH GM121455 对 S.A.G 的最新支持。有关 嗜粪芽孢杆菌 的更多详细信息，请访问目前正在建设的 Bradysia （Sciara） 种群中心网站（https://sites.brown.edu/sciara/ 和 https://sciara.wi.mit.edu）。

Bradysia coprophila Lab Protocols and Chromosomal Research

作者没有需要声明的利益冲突。

这里介绍的嗜 粪芽孢杆菌 饲养方案对于希望在实验室中饲养这种生物体进行实验以深入研究其独特生物学特征的科学家非常有用。在梅斯实验室中，使用酵母和蘑菇粉撒在琼脂基上以维持嗜 粪芽孢杆菌29 的饲养方法的初步描述用于饲养 14 种不同种类的 Sciarid 果蝇5。随后，观察到添加荨麻和/或菠菜粉进一步增加了 嗜酸芽孢杆菌 （Gabrusewycz-Garcia，个人交流）的活力。这些方法已经成功地维持了 Sciaridae 家族内的相关物种，包括目前正在培养的 Bradysia impatiens 和 Lycoriella ingenua （Robert Baird，个人通信）。
已经尝试了其他方法（例如下面描述的替代喂养方法）来培养 嗜粪芽孢杆菌，但此处描述的方案已经过优化，以具有最有利的幼虫单位琼脂表面积比例，以获得最肥沃的脂肪成虫并最大限度地减少霉菌的生长。为了扩大规模，可以按照上述方案 2 中的说明在玻璃瓶中进行质量配接。或者，可以将一些 （2-4） 成年雌性与两倍数量的成年雄性一起放入一个培养瓶中，例如用于饲养 果蝇 的培养瓶（一次性 6 盎司 = 177.4 mL 方底 果蝇 聚丙烯瓶）。在这两种情况下，研究人员都必须完全确信培养瓶仅包含所有女性生产者或所有男性生产者母亲。
只喂幼虫，因为蛹和成虫不吃东西。如果幼虫已经变成蛹，请不要喂食小瓶（一个迹象是第一批早出的成蝇出现时）。一旦成虫关闭，如果可用，将小瓶放入较冷的培养箱（例如，16 °C）中，因为这将使成虫活得更久。每周喂食 3 次（例如，周一、周三、周五），随着幼虫年龄的增长，每瓶的喂食量增加。慷慨地喂养，你会得到肥大可育的成年人的回报。但是，如果您喂食过多，会出现白色霉菌，这是减少您存放在小瓶中的食物量的迹象。此外，如果你喂得太多，琼脂顶部会形成一层厚厚的食物垫，使成虫更难出现（你可以用镊子去除垫子，但要注意不要用垫子把幼虫带出来——最好根本不这样做）。幼虫很少（标记为"很少"）的小瓶需要更少的食物。如果你喂得太少，幼虫会爬上小瓶的壁寻找食物。喂养不足的幼虫会导致小成虫的生育能力较差。
替代饲喂方法 已经尝试了多种方法，在幼虫阶段只喂养幼虫一次，而不是每周 3 次。B. coprophila 不会在 果蝇 风格的食物上生长。John Urban（个人通信）尝试将 嗜酸芽孢杆菌 食物与琼脂混合，但滋生了太多霉菌。他发现，混合添加两种霉菌抑制剂（tegosept 和丙酸）并分别尝试几种不同的浓度，在抑制霉菌的水平上，都对 嗜霉芽孢杆菌 有毒。琼脂的 pH 值应为 6-7（中性），因为 嗜酸芽孢杆菌 在酸性 pH 值（如丙酸）下会生病。或者，为了避免每周三次喂食，他尝试使用抹刀或没有针头的注射器，在交配后一周（即，大约幼虫开始出现的时间）将浓稠的酵母糊（Red Star 活性干酵母与少许蒸馏水混合以润湿）作为一团分配到每个小瓶的琼脂顶部。
避免每周喂食三次的另一种方法是在每个小瓶中加入活的真菌培养物。Bath 和 Sponsler37 报道了具有 Sabouraud 培养基的倾斜琼脂表面应用 Chaetoconidia 属（最好）或 Baplosporangia 或 Xllescheria 属的真菌培养物进行条纹。这种真菌是在引入嗜 粪芽孢杆菌 前几天到一周生长的。此后不需要喂食。Ellen Rasch （个人通信） 也采用了这种方法的变体。在我们手中，用这种方法小瓶太湿，幼虫溺水，但可以再次尝试以优化相对于含有活真菌的小瓶的幼虫数量。
Arthur Forer（个人通信）在以与鹤蝇相同的方式饲养 B. coprophila 方面取得了一些成功38.通过这种方法，蛹被饲养在潮湿的 纸浆上。随后，成虫交配，卵沉积在新鲜潮湿的 纸浆上。所得幼虫被保存在培养皿中的 纸浆 上，每周两次用荨麻叶粉喂养。将蛹放入笼子中以重复该循环。
Yukiko Yamashita（个人通信）尝试在土壤上饲养 B. coprophila，但未成功，模仿它们在自然界中高湿度的盆栽植物和温室中的条件。但是，当湿度水平升高时，霉菌会成为一个问题。尽管如此，潮湿的土壤已经成功地用于在装有潮湿土壤的塑料盒中培养假石蒜属（以前的 Bradysia）hygida 幼虫;它们以分解的 Ilex paraguariensis 叶为食，在幼虫晚期补充 1.2% 酵母提取物、1.4% 玉米淀粉、0.8% 燕麦片粉、1.2% 琼脂12。同样，潮湿的土壤可以用带有碎芸豆的潮湿泥炭藓代替，以培养 Sciarid 苍蝇39,40。
还采用了其他方法来维持 Bradysia 的实验室培养:（i） 添加酵母和干血肥的蒸压马铃薯41;（ii） 粪污 42,43,44 可添加干血 45;（iii） 装有化妆棉和装有大豆粉的湿纸巾的塑料容器46.
螨 螨虫可以从果蝇转移到嗜果芽孢杆菌。为了尽量减少这种情况，最好将嗜黑芽孢杆菌放在单独的培养箱或房间里，不要靠近果蝇库存。此外，在处理果蝇之前，请在当天早些时候进行任何嗜果蝇维护工作。螨虫也可以从室内植物转移到嗜红芽孢杆菌，因此不要将植物与嗜红芽孢杆菌放在同一个房间里。如果螨虫侵入小瓶，它们可以被视为在 B. coprophila 身体上爬行的白色小球形生物。用于消灭果蝇中螨虫的化学处理不能用于嗜果蝇，因为化学物质会杀死嗜果蝇（嗜果蝇也对苯酚等有机烟雾敏感）。去除嗜粪芽孢杆菌原液中螨虫的唯一治疗方法是手动将胚胎收集在琼脂平板上，检查每个胚胎是否不含螨虫，然后使用细画笔将它们转移到新鲜的琼脂瓶中。棉布填充的纱布塞和醋酸纤维素泡沫绒毛（用于果蝇聚丙烯样品瓶）都有助于防止螨虫进入样品瓶。
饲养方案的有用性 此处描述的协议将使不断增长的科学家社区能够将 嗜粪芽孢杆菌 饲养为新的/旧的新兴模式生物，以研究其独特的生物学特征。鼓励新的实验室小组加入不断壮大的社区，以维护和研究 Bradysia （Sciara） 的独特生物学特征。

要全面了解生物学原理，需要研究跨越生命之树的许多不同生物。尽管到 19世纪末 描述了广泛的生物体，但到 20世纪 中叶，实验研究仅限于少数不到十几种模式生物。随着基因组时代的到来以及对生命之树1 中所有物种的基因组进行测序的目标，我们现在能够扩大用于实验室实验的生物类型，并利用其多样性的优势。这种对新兴模式生物进行实验的扩展具有能够在实验室中维护它们的先决条件。在这里，描述了饲养一种这种新兴新/旧模式生物的方案。
地球上的大部分动物生命是由 Insects2 的四种超级辐射构成的。在昆虫中，大约有 158,000 种双翅目（真苍蝇）3，其中大约 3000 种属于 Sciaridae（黑真菌蚊蚋）4。果蝇果蝇是双翅目果蝇中研究最彻底的。低级双翅目果蝇 （Nematocera），Bradysia（以前称为 Sciara）coprophila，在 2 亿年前与果蝇分化，果蝇是一种"高级双翅目"苍蝇 （Brachycera）。因此，嗜黑双歧杆菌处于与黑腹果蝇比较研究的有利分类位置（图 1）。此外，嗜粪芽孢杆菌具有许多独特的生物学特征，本身就值得研究 5,6,7。其中许多特征违反了 DNA 恒定性规则，在该规则中，生物体的所有细胞都具有相同的 DNA 含量。在 B. coprophila 中，（i） 在雄性减数分裂 I 中，父系基因组在单极纺锤体上被消除;（ii） 雄性减数分裂 II 中存在 X 二元组不分离;（iii） 从体细胞中消除生殖系限制性 （L） 染色体;（iv） 根据个体的性别，在早期胚胎中消除一条或两条 X 染色体。用于区分母系和父系同源物的染色体印记首先在 B. coprophila 中发现，并在许多这些染色体消除事件中发挥作用。除了染色体消除外，DNA 恒定性的另一种旁路是通过幼虫唾液腺多线染色体中 DNA 泡芙位点的发育调节、基因座特异性 DNA 扩增发生的。这些独特特征的研究需要对嗜酸芽孢杆菌进行实验室维护;这里介绍其饲养的详细信息，以促进此类研究。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66751/66751fig01v3.jpg"> 图 1: Bradysia （Sciara） coprophila 的系统发育。 流行的模式生物以蓝色字体表示，它们的分类顺序以红色字体表示。 Bradysia 和其他 Sciarid 真菌蚊蚋以及蚊子是低级双翅目苍蝇（以前是 Nematocera 亚目），而 果蝇 物种是高等双翅目蝇（亚目:Brachysera）。图左侧的信息来自 Misof 等人 33;右侧的信息来自 Bertone 等人 34 和 Wiegmann 等人 2。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
以前，Sciara 属在所有真核生物中拥有最多的物种 （700），促使 Steffan 将它们细分为8 种。随后，Shin 提议将 Sciaridae 科细分为 Sciarinae 亚科（有六个属，包括 Sciara、Trichosia 和 Leptosciarella）、Megalosphyinae 亚科（包括 Bradysia 属）和其他三个组（包括 Pseudolycoriella）9。近年来，几个小组进一步研究了 Sciaridae 的系统发育 9,10,11。在过去的几十年里，Sciaridae 家族中许多生物的名称发生了变化12.尽管跨越一个多世纪的大多数文献都将我们研究的生物体称为 Sciara coprophila，但它目前的分类学名称现在是 Bradysia coprophila（同义 Bradysia tilicola 和其他同义词）10。它们分布于世界各地，通常被称为真菌蚊蚋，因为它们吃蘑菇和其他真菌。它们于 1804 年由欧洲的 Meigen13 首次描述，随后由北美的 Johannsen 14,15 描述。B. coprophila 是在冷泉港实验室收集的，实验室库存是由 Charles Metz 在 1900 年代初期建立的，当时他还是哥伦比亚大学的研究生，师从托马斯·亨特·摩根 （Thomas Hunt Morgan）。因此，目前的种群反映了一个世纪的近亲繁殖。同样，Helen Crouse（她与 Barbara McClintock 一起完成博士工作）数十年的细胞遗传学研究进一步阐明了嗜粪芽孢杆菌的生物学特性。
在 1930 年代，Bradysia （Sciara） 与黑腹果蝇竞争作为遗传研究的模型系统。尽管具有许多独特的生物学特征，但嗜黑腹果蝇 （B. coprophila） 作为一种流行的模式生物被嗜黑腹果蝇所掩盖，因为遗传研究需要辐射诱导的表型突变，并且在后者中更容易实现，尽管嗜黑果嗜血杆菌对 γ 照射的抵抗力仅比嗜黑腹果蝇 （D. melanogaster） 略高 16。在基因组学的现代时代，这不再是一个问题。由于嗜粪芽孢杆菌的基因组序列 17,18,19（Urban、Gerbi 和 Spradling，数据未显示）和转化方法20,21（Yamamoto 和 Gerbi，数据未显示）最近已经可用，现在是时候将其用作新的/旧的新兴模型系统了，正如越来越多的科学家社区所看到的那样，他们已经将其用于研究。本文介绍了其实验室维护的程序。
B. coprophila 缺乏 Y 染色体，后代的性别由母亲决定。具有 X'（"X-prime"）染色体且具有长近着丝粒倒置的女性将仅生育女儿，而标准（非倒置）X 染色体纯合子的女性将仅生育5 个儿子（图 2）。X'19 号染色体的序列信息是可用的，但 X' 染色体如何决定后代将是女性的分子机制仍有待阐明。雄性从来没有 X' 染色体，受精后，雌性是 X'X（X' 的杂合子）或 XX。成年 X'X 雌性可以通过翅膀上的表型标记与 XX 雌性区分开来（图 3）。X'X 雌性（将只生女儿）可以通过 X' 上占主导地位的波浪形 （W） 翅标来识别（如 HoLo2 库存）22。或者，XX 雌性（将只有儿子）可以通过 X 上的隐性娇小 （p） 翅标来识别，如 91S 股票23。在这种情况下，X'Xp 雌性将具有全长（不是娇小）的翅膀，并且只有女儿。原种 6980 在 X 染色体上带有一个用于肿胀 （sw） 静脉24 的隐性标记，以及 X' 上的主要标记 Wavy，允许两个标记用于选择杂交。Wavy 的表达程度可能会有所不同，并且在食物受限或温度过高的过度拥挤的小瓶中似乎较弱。如果将幼虫保存在冷藏室 （4°-8 °C） 而不是通常的 21 °C 中，则波浪翼表型非常强。 虽然隐性娇小翅膀标记不是可变的并且很容易识别，但 91S 股票的使用频率较低，因为它们不如 HoLo2 股票健康。这里介绍了嗜粪菌 B. coprophila 的交配方案（图 2），并详细描述了 HoLo2、7298 和 W14 种群（补充文件 1）、91S 种群（补充文件 1）、6980 种群（补充文件 1）和易位种群（补充文件 1）。易位种群已不复存在;它们是异色粒 （H1、H2 和 H3） 在包含核糖体 RNA 基因 25,26,27 的短臂上的互易位。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66751/66751fig02.jpg"> 图 2: 嗜粪芽孢杆菌的交配方案。 这种生物体没有 Y 染色体（雄性胞体只有一个 X）;母亲决定后代的性别。XX 个母亲只有儿子，X'X 个女性只有女儿。与 X 染色体相比，X' 染色体具有较长的近着丝粒倒置。X （或 X'） 染色体的父系或母系谱系在此图中分别用蓝色或红色表示。精子是常染色体的单倍体，但由于减数分裂 II 中的不分离，精子具有两个 X 染色体副本。早期胚胎的体细胞谱系会分别消除一个或两个父系衍生的 X 拷贝，如果它们是女性或男性。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66751/66751fig03.jpg"> 图 3: 嗜红芽孢杆菌的翅膀表型。 成年雌性果蝇具有各种翅膀表型:（A） 直翅 （XX），（B） 波浪形翅膀 （X'WX），（C） 极端波浪形翅膀 （X'WX） 表型，在将幼虫储存在寒冷的roo m 中后外观干瘪，（D） 娇小的翅膀 （XpXp） 退化样，（E） 直翅，野生型 （XX） 且没有肿胀的叶脉， （F） 静脉肿胀的直翅 （XswXsw），其中小气泡（黑色箭头）出现在翅膀的上边缘和/或两个翅膀的尖端附近，（G） 肿胀的极端例子，一个或两个翅膀上出现水泡（白色箭头）。雄性没有 X' 染色体，因此永远不会有波浪形的翅膀，但它们分别在 91S 或 6980 库存中有娇小或肿胀的翅膀。比例尺 = 1 毫米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig03large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
存货维护的目标是进行杂交，其中一半的杂交来自产母的母牛，一半的杂交产母的母牛，以便在下一代中产雌母的成年母牛和成年雄牛的数量相等，用于后续的杂交。然而，这也涉及计划，因为雄性的生命周期比雌性短，而且成年雄性比成年雌性早一周出现。大自然通过让雄性胚胎在同一日期从杂交中从雌性幼虫中出来 1-2 天作为幼虫出现来适应这种性别之间的异步。然而，为了确保雄性和雌性成虫同时可用于实验室杂交，通过将带有雌性幼虫的小瓶放在室温而不是 21 °C 或将带有雄性幼虫的小瓶放置在稍低的温度（例如，16 °C）中，可以在一定程度上加快雌性幼虫的发育。另一种更万无一失的方法是在周一与女性生产者母亲进行杂交，在同一周的周五与男性生产者母亲进行杂交。最简单的方法，也是我们采用的，是在每周的同一天与女性生产者和男性生产者的母亲进行杂交，并在每周的同一天进行杂交。在这种方法中，第 1 周杂交的成年雌性可以与第 2 周杂交的成年雄配。
在 21 °C 下饲养时，雌性 嗜粪芽孢杆菌 的生命周期为 5 周（表 1）。在较低的温度下或如果它们喂养不足，它们的生命周期长度会更长一些。雄性嗜 粪 菌的生命周期为 ~4-4.5 周，因为它们比雌性早 0.5-1 周化蛹。每个幼虫龄期的末端以角质层脱落为标志，这是由类固醇激素蜕皮激素水平的爆发触发的。与具有 3 个幼虫龄的 D. melanogaster 不同， B. coprophila 有 4 个幼虫龄。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>发育阶段</td>
			<td>配接后天数 （dpm）</td>
			<td>阶段长度（天）</td>
		</tr><tr><td>产蛋</td>
			<td>1-2</td>
			<td></td>
		</tr><tr><td>胚胎</td>
			<td>1-2 至 7-8</td>
			<td>~7 天</td>
		</tr><tr><td>幼虫</td>
			<td></td>
			<td></td>
		</tr><tr><td>幼虫龄 1、2 和 3</td>
			<td>7-8 至 16-19</td>
			<td>~10</td>
		</tr><tr><td>第 4 幼虫龄前眼点</td>
			<td>16-19 至 21-24</td>
			<td>5</td>
		</tr><tr><td>第 4 幼虫龄眼点期</td>
			<td>21-24 至 25-28</td>
			<td>4</td>
		</tr><tr><td>蛹</td>
			<td>25-28 至 30-33</td>
			<td>5</td>
		</tr><tr><td>成人</td>
			<td colspan="2">如果交配，在 21 °C 下可存活 1-2 天，如果不交配，在 16 °C 下可存活 2-3 周。</td>
		</tr></tbody></table>表 1:21 °C 下 雌性嗜红芽孢杆菌 的生命周期。
B. coprophila 可以保存在 15 °C-25 °C 范围内的任何温度，在较冷的温度下发育进展较慢。这种昆虫喜欢潮湿的环境（在室内植物或蘑菇床的土壤中发现），因此我们在培养箱中放置了一个装有去离子水的烧杯。 嗜酸芽孢杆菌 可以在室温下保存在金属面包盒中，该面包盒带有松散的盖子并装有水杯，但它们在 37 °C28 时会发生热休克，这在炎热的气候下是很危险的。Michael Ashburner 和其他人曾尝试将 黑腹果蝇 冷藏，以减少库存所需的时间，但收效甚微。相比之下， 嗜粪芽孢杆菌 的一个主要优点是，带有中期幼虫的小瓶可以在冷藏室 （4-8 °C） 的开放式架子上储存长达 3 个月，只需每月饲喂一次。它们在寒冷中发育极其缓慢，直到蛹期，当小瓶恢复到 21 °C 时，它们会成为可育的成虫。 据推测，这模仿了它们在野外越冬。这种寒冷诱导的发育停滞可能与中期 嗜粪芽孢杆菌 幼虫16 的伽马射线照射后看到的情况相当，但在已经过了正常发育进程不归路的晚期幼虫中看不到发育停滞。

<table><tbody><tr><td>Agar (bacteriological)</td><td>U.S. Biological</td><td>A0930</td><td>https://www.usbio.net;&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; FlyStuff Foot Pedal</td><td>Genesee Scientific</td><td>59-121</td><td></td></tr><tr><td>CO2 FlyStuff Blowgun</td><td>Genesee Scientific</td><td>54-104</td><td></td></tr><tr><td>CO2 FlyStuff UltimaterFlypad</td><td>Genesee Scientific</td><td>59-172</td><td>https://www.geneseesci.com</td></tr><tr><td>Ether fume hood</td><td>Labconco&amp;nbsp;</td><td>3955220</td><td>Sits on top of lab bench</td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>Filter replacement&amp;nbsp; cat # 6961300</td></tr><tr><td>Food: Brewer&amp;rsquo;s Yeast Powder</td><td>Solgar</td><td>&amp;nbsp; &amp;nbsp;</td><td>Obtain from Amazon or health food store</td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>https://www.solgar.com;</td></tr><tr><td>Food: Nettle Powder&amp;nbsp; (pesticide free)</td><td>Starwest Botanicals</td><td>209460-51</td><td></td></tr><tr><td>Food: Shitake Mushrooms (pesticide free)</td><td>Starwest Botanicals</td><td>202127-5</td><td>https://www.starwest-botanicals.com;</td></tr><tr><td>Food: Spinach Powder ( pesticide free)</td><td>Starwest Botanicals</td><td>209583-5</td><td></td></tr><tr><td>Food: Straw (pesticide free )</td><td>Starwest Botanicals</td><td>209465-3</td><td></td></tr><tr><td>Jar: clear glass, polypropylene lid</td><td>Fisher Scientific:</td><td>FB02911765</td><td>73 mm dia, 89 mm ht (240 ml)&lt;br/&gt; https://www.fishersci.com;</td></tr><tr><td>Needle Probe, wooden handle</td><td>US Geo Supply Inc</td><td>SKU: 4190</td><td>5.75&amp;rdquo; long probe,&lt;br/&gt; stainless steel needle&lt;br/&gt; https://usgeosupply.com; (970)-434-3708</td></tr><tr><td>Vials: glass, preferred:</td><td>Wilmad LabGlass</td><td></td><td></td></tr><tr><td>Wilmad-glass custom vials</td><td></td><td></td><td>28-33 mm inner dia, 33 mm outer dia, 9.5 cm ht&lt;br/&gt; Wilmad: https://www.SP-WilmadLabglass.com</td></tr><tr><td>Vials: glass (cheaper and ok)&amp;nbsp;</td><td>Fisher Scientific</td><td>03-339-26H</td><td>29 mm outer dia, 9.5 cm h&lt;br/&gt; &amp;nbsp;https://www.fishersci.com;&amp;nbsp;</td></tr><tr><td>Vials: glass (a bit narrow)&amp;nbsp;</td><td>Genesee Scientific&amp;nbsp;</td><td>32-201</td><td>24.5 mm outer dia,9.5 cm h&lt;br/&gt; thttps://www.geneseesci.com</td></tr><tr><td>Vials: polypropylene&amp;nbsp;</td><td>Genesee Scientific</td><td>32-114</td><td>28.5 mm outer dia,9.5 cm ht</td></tr><tr><td>Vial Plugs</td><td></td><td></td><td></td></tr><tr><td>roll of non-absorbent cotton</td><td>Fisher Scientific</td><td>22-456881</td><td></td></tr><tr><td>cheesecloth</td><td>Fisher Scientific</td><td>22-055053</td><td>https://www.fishersci.com;</td></tr></tbody></table>

laboratory maintenance of the lower dipteran fly bradysia (sciara) coprophila: a new/old emerging model organism

低双翅目蝇 Bradysia （Sciara） coprophila 的实验室种群已经维持了一个多世纪。此处介绍了 嗜粪芽孢杆菌 的实验室维护方案。这些方案将有助于迅速增加的研究 嗜盲芽孢杆菌 的实验室利用其独特的生物学特征，其中包括 （1） 雄性减数分裂 I 中的单极纺锤体;（2） 雄性减数分裂 II 中 X 二元组不分离;（3） 染色体印记以区分母系和父系同源物;（4） 生殖系限制性 （L） 染色体;（5） 染色体消除（雄性减数分裂 I 中的父系染色体;早期胚胎中的一到两条 X 染色体;来自早期胚胎胞体的 L 染色体）;（6） 母亲决定性别（没有 Y 染色体）;（7） 幼虫唾液腺多线染色体中 DNA 泡芙位点的发育调节 DNA 扩增。 
现在，通过使用 嗜粪芽孢杆菌 基因组测序和组装的最新进展以及基因组工程转化方法的开发，可以探索染色体力学的这些独特特征。使用 B. coprophila 进行研究的不断增长的科学界将受益于此处描述的苍蝇交配方案（仅生儿子或独生女的母亲的表型标记;生化实验的大规模交配细节）、检查胚胎孵化、喂养幼虫以及对其饲养的其他评论。

此处描述的方案已被证明在饲养 嗜红芽孢杆菌方面取得了成功。当选择最近封闭的脂肪成虫进行交配时（图 4），超过 90% 的杂交可以生育并产生后代。不同的种群确实会有所不同（表 2）。7298 原种（带有 Wavy 标记的 X' 染色体）是最健康的原种，但经历了一段下降期，这显然是由于 DNA 移动元件的激活产生了基因组重排32。HoLo2 原液代表源自 7298 的健康菌株，其中基因组重排显然已经稳定，并且它已经取代了原液中心的亲本 7298 原液。HoLo2 原液是用于对 嗜红芽孢杆菌 基因组进行测序的原液，也是各种实验室小组使用最广泛的原液。最近，HoLo2 果蝇的 CRISPR 诱变已被用于创建具有白色眼睛表型的 W14 原液，用于用荧光眼标志物进行转化（Yamamoto 和 Gerbi，数据未显示）。W14 菌株非常坚固。6980 枪托（波浪形机翼和肿胀的静脉标记）的强度稍逊一筹，而 91S 枪托（小翼标记）的强度甚至更低。
成功的杂交会产生胚胎（图 5）。胚胎在第 7 至第 9次 卵裂分裂时去除印记的父系 X 染色体。此外，在胚胎的第 5 至 第 6次 卵裂分裂时，生殖系限制的 L 染色体从体细胞谱系中被消除。胚胎以幼虫的形式出现，不应与也可能存在的霉菌混淆（图 6）。眼点（对成虫眼睛的眼球）出现在第 4 龄 幼虫的后半部分（图 7）。眼点的大小为 DNA puff 扩增的开始和进展提供了方便的表型标志物，这是已知的仅有的两个自然发生的发育调节位点特异性染色体内 DNA（基因）扩增的例子之一。随后，蛹发育，充满眼睛的色素量可以作为精子发生中减数分裂 I 和 II 的发育标志物（图 8），其在这些分裂中具有独特的染色体行为。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66751/66751fig04.jpg"> 图 4: 嗜红芽 孢杆菌成蝇的交配。 上面板显示了 HoLo2 种群中的三种成年苍蝇:波浪翅膀的雌性产卵母（X'WX 成年雌性）、直翅的雄性产卵母（XX 成年雌性）和直翅的雄性（X0 成年雄性）。注意雌蝇后端的尖产卵器和雄蝇后端的钩状扣环。下面板显示了雄性和雌配，雄性扣子抓住了雌性的产卵器。精子将储存在女性的精子中，并在卵子排放到外部时使卵子受精。成虫的长度为 2.0 毫米（雄性），2.5 毫米（雌性）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66751/66751fig05v2.jpg"> 图 5:嗜粪芽孢杆菌胚胎。左上面板是在解剖显微镜中使用标准光的胚胎视图;合胞体细胞质中的细胞核显示为黑点。右图使用荧光显微镜观察碘化丙啶染色的胚胎细胞核。胚胎的平均长度为 200 微米，平均宽度为 150 微米。生殖细胞的细胞核聚集在胚胎的后极，如第 6 周期（胚胎向前倾斜）和第 7-9 周期所示，之后它们散布着体细胞核。体细胞谱系中的 L 染色体消除发生在卵裂分 5 或 6 中;体细胞谱系中的 X 染色体消除发生在第 7、8或 9裂解分裂。细胞化发生在第 11 周期的间期。左表显示了 22 °C 时每个分割周期的平均时间长度。 左侧的桌子和右侧的面板经 de Saint Phalle 和 Sullivan35 许可改编。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66751/66751fig06.jpg"> 图 6: 嗜粪芽孢杆菌 胚胎以幼虫的形式出现。 在产卵后死亡的成年雌性产卵器附近可以看到一簇胚胎（蓝色粗箭头）。产卵一周后，胚胎变成年轻的幼虫，其中几只幼虫用黑色箭头表示。新出现的幼虫在前端有一个黑色的下巴和一个半透明的身体。它们在琼脂表面的顶部移动，不应与不移动的霉菌混淆。插图显示一些霉菌，有一根白色细丝（红色箭头）和尖端的黑色孢子（绿色箭头），比新出现的幼虫略小。比例尺 = 1 毫米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig06large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66751/66751fig07.jpg"> 图 7: B. coprophila 幼虫的眼点阶段。 眼点形成于幼虫的前部，就在下颌的后面，由数量增加的色素颗粒组成。眼点是成人眼睛的眼罩。上面板显示了用解剖显微镜观察的幼虫;下面板是使用相差显微镜在显微镜载玻片上观察幼虫的放大视图，上面有一滴蒸馏水和盖玻片。眼点阶段的命名是根据 Gabrusewycz-Garcia30 的，其中颗粒数计算在最长的行中（例如，12），并记录除最长行之外的额外行数（例如，眼点阶段 6x6）。唾液腺多线染色体中位点特异性 DNA 扩增的开始始于眼点阶段 10x5，并在 14x7 时完成，此时基因座出现转录爆发并扩增 DNA 泡泡31。在随后的边缘眼/下颌阶段，眼点颗粒开始合并，并横向远离中线;幼虫体的长度缩短。此外，DNA 泡泡在这个阶段凝结。在 21 °C 下穿过每个眼点阶段大约需要一天时间。比例尺 = 1 毫米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig07large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66751/66751fig08.jpg"> 图 8: B. coprophila 蛹的发育。 在化蛹过程中，除神经系统外，所有幼虫组织都会组织裂解，并被假想盘细胞分裂产生的成体组织所取代。体色从白色变为棕褐色，再变为棕色，再变为黑色。色素逐渐填充蛹眼;减数分裂 I 和 II 发生在雄性蛹中，眼睛 1/4 至 1/2 充满色素36。比例尺 = 1 毫米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/66751fig08large.jpg" target="_blank">请点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>股票名称</td>
			<td>标记</td>
			<td>生育率</td>
			<td>评论</td>
		</tr><tr><td>7298</td>
			<td>波浪形 （W） 机翼</td>
			<td>~75%</td>
			<td></td>
		</tr><tr><td>霍洛2</td>
			<td>波浪形 （W） 机翼</td>
			<td>~90%</td>
			<td>源自 7298</td>
		</tr><tr><td>周14</td>
			<td>波浪形 （W） 翼;白色的眼睛</td>
			<td>~95%</td>
			<td>源自 HoLo2</td>
		</tr><tr><td>6980</td>
			<td>波浪形 （W） 翼;肿胀 （SW） 静脉</td>
			<td>~65%</td>
			<td></td>
		</tr><tr><td>91 年代</td>
			<td>弱的波浪形 （W） 翼;娇小 （P） 翅膀</td>
			<td>~50%</td>
			<td>Wavy 标记被引入十字架以拯救 91S</td>
		</tr></tbody></table>表 2:Bradysia （Sciara） coprophila 的种群。Gerbi6 的表 1 列出了这些标记和其他不再存在的标记。X27 着丝粒末端的 5 次易位 （T1、T23、T29、T32、T70） 总结在 Gerbi6 的图 8 中，但已不存在。
补充文件 1:HoLo2（以及 7298 和 W14）杂交、91S 杂交和救援、6980 杂交、易位杂交。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66751/Supplemental%20Materials.zip" target="_blank">请点击此处下载此文件。</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring Bradysia coprophila's Unique Biology: A Guide to Laboratory Maintenance at JoVE.com

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

本文概述了低双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护（包括交配和喂养）。

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

我们保留了低双翅目苍蝇 Bradysia，也称为 Sciara coprophila，以研究其一些独特的生物学特性，包括减数分裂中的单极纺锤体和 x 二元不分离、染色体印记、种系特异性染色体、染色体消除、母系性别决定和污染性染色体 DNA 泡泡中的 DNA 扩增。在我们的实验室中，Yutaka Yamamoto 开发了 Bradysia coprophila 基因组转化的方法。此外，这种生物体的转录组已经可用，其基因组已由 John Urban 测序和组装。这些突破使探索 Bradysia coprophila 的独特生物学成为可能。我们鼓励您加入不断壮大的科学家社区，以解决 Bradysia coprophila 的独特特性。您的结果将阐明对细胞和发育生物学具有根本重要性的分子机制。此处介绍的饲养方案将作为在您自己的实验室中维护 Bradysia coprophila 的教程。

laboratory-maintenance-lower-dipteran-fly-bradysia-sciara-coprophila

Universidad San Sebastian

Research

JoVE Journal

Biology

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

899 次观看.  Brown University. 我们保留了低双翅目苍蝇 Bradysia，也称为 Sciara coprophila，以研究其一些独特的生物学特性，包括减数分裂中的单极纺锤体和 x 二元不分离、染色体印记、种系特异性染色体、染色体消除、母系性别决定和污染性染色体 DNA 泡泡中的 DNA 扩增。在我们的实验室中，Yutaka Yamamoto 开发了 Bradysia coprophila 基因组转化的方法。此外，这种生物体的转录组已经可用，其基因组已由 John Urban ...

视频: Author Spotlight: Exploring Bradysia coprophila's Unique Biology – A Guide to Laboratory Maintenance

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

I am Es.In MindUP, we study biology of human pathogens in non hosts environments. Many human pathogens after exiting their human host have to survive in the environment before infecting another host. So our goal is to better understand processes that control survival of human pathogens in the environment.And our goal is to then better understand transmission dynamics of infectious agents. So we are using Vibrio cholera, the cost of agent of diarrhea, disease, cholera as a model organism. Vibrio cholera is a facultative human pathogen and it has an aquatic lifecycle.Therefore, it provides us great opportunity to study the survival of human pathogen in a natural environment. There are many tools to study this organism. Its genome is completely sequenced and there are micro array representing every gene in the genome.And furthermore, there are many tools that we can manipulate the genome of this organism. My research program takes advantage of these tools and integrates them to study aquatic lifecycle of cholera. So in our experiments, we try to simulate the aquatic environments that organism lives in.And then we would like to know how does this organism so senses fluctuations in its environment with respect to nutrient load, temperature, salinity, or other biological stressors such as pages and tzo? So in aquatic environment, RIA cholera is found either in a free living form or in a surface attached form. And the surface attached forms are known as biofilms, which are simply composed of microorganisms and their extra polymeric substances, which can be exo polysaccharide and proteins and sometimes DNA.And this matrix acts as a glue and hold the organism together. And when grown in biofilm mode, these organisms are actually resistant to fluctuating parameters of their environments and they can be better protected from, for example, phage and proteasome probations. And these are factors that are controlling abundance of microorganisms in the environment.Some of the recent techno technological advance that helped us in our studies are the completion of the genome sequence of the organism and development of microray technology. Using these technologies, we can determine at the whole genome level how this organism responds to major fluctuations in its environment such as nutrient deprivation or changes in temperature. Or one of our major goal is to integrate our whole genome expression studies to a network analysis.So we would like to identify genetic regulatory network that controls response of to fluctuating environmental parameters. We would like to be able to model behavior of organism in the environment. And also we still do not know when in the environment with whom Vibra Cide interacts with.We do not know much about ecology of this organism, and we also do not know relevance of the genes and processes we identified to be important for survival of this organism in the laboratory to the real world. No are the genes that we identified express when Ria cholera is present in natural environment. We also would like to be able to identify and culture organisms that Vibrio Cora interacts with because in the environment, Vibrio Cora is a part of the complex microbial community.So I think understanding biology of Vibrio Cora in natural environment would be a, it's a challenging and it's critical for us to understand biology of the organism. Within the next five years, I hope that we would be able to better characterize op polysaccharide components of biofilms. For example, in Vibrio cholera, we still do not know structure of the op polysaccharide that is holding the cells together.We also do not know all the components of biofilm metrics. Eventually I think it be great to be able to quantify components of the biofilm metrics in natural biofilms of these organisms. I think another major challenge is to study, be OC in natural environment.And I hope that we would be able to take advantage of comparative genomic studies and combine whole genomic expression profiling comparative genomics to better understand evolution of IBR cholera as a pathogen.

霍乱弧菌：模式生物研究细菌的发病机制 - 访

科研

生物学

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

生物化学

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 ...

环境科学

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

生存时间的测量<em&gt;臂</em&gt;轮虫:产妇条件同步

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

维持 双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

在实验室和田间条件下培养 Crepidula fornicata 的幼虫

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded veliger larvae of this species were collected by siphoning onto a sieve after natural release by adults, distributed into the culture at a density of 200/L, and fed with Isochrysis galbana (strain T-ISO) at 1 x 105 cells/mL. Shell growth and acquisition of competence for metamorphosis were documented for sibling larvae reared in ventilated 800 mL cultures designed for equilibration to ambient air or to defined atmospheric gas mixtures. Contrasting with these laboratory culture conditions; growth and competence data were also collected for larvae reared in a 15 L flow-through ambient seawater mesocosm located in a field population of reproductive adults. Growth rates and timing of metamorphic competence in the laboratory cultures were similar to those reported in previously published studies. Larvae reared in the field mesocosm grew much faster and metamorphosed sooner than reported for any laboratory studies. Together, these methods are suited for exploring larval development under predetermined controlled conditions in the laboratory as well as under naturally occurring conditions in the field.

作者聚焦：弥合幼虫生态学研究中现场观察和实验室操作之间的差距

The paper presents methods for larval culture of the gastropod Crepidula fornicata in a small-volume laboratory-scale system and in an ambient-seawater mesocosm system that can be deployed in the field.

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

副本

摘要

探索更多视频

霍乱弧菌：模式生物研究细菌的发病机制 - 访

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

藤壶Balanus improvisus作为海洋模型的培养和基因表达

作为一个型号的遗传生物鳉胚胎显微注射

定期保养和维护的斑马鱼（

从环境中自然产生的趋磁细菌的采集，分离和富集

一个单细胞消融从甲壳动物端足八细胞胚胎

生存时间的测量

维持双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

副本

摘要

探索更多视频

霍乱弧菌：模式生物研究细菌的发病机制 - 访

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

藤壶Balanus improvisus作为海洋模型的培养和基因表达

作为一个型号的遗传生物鳉胚胎显微注射

定期保养和维护的斑马鱼（

从环境中自然产生的趋磁细菌的采集，分离和富集

一个单细胞消融从甲壳动物端足八细胞胚胎

生存时间的测量

维持 双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

维持双斑蝽螈的实验室培养物，这是一种用于昆虫农业和无脊椎动物生理学的多功能正翅目动物模型