用细胞遗传学技术测定芽孢杆菌的受精率
Summary
我们提出了一个简单的细胞遗传学技术, 利用 4 ‘, 6-二胺-2-苯基苯二酚 (dapi) 来确定施肥率和主要性别比例的单倍体入侵害虫杆菌。
Abstract
少数吸刺的白蝇是世界上最具破坏性的陆地害虫之一, 因为它们对作物造成了损害, 并使它们成为媒介。尽管对这些物种在不同环境中的生物学进行了大量研究, 但一个关键的生活史参数—-后代性别比率—-却很少受到关注, 但对预测种群动态很重要。这种单倍体昆虫的主要性别比 (产卵性别比) 从未报道过, 但通过确定单倍体昆虫的受精率可以找到。该技术包括用漂白剂对鸡蛋进行脱色, 采取一系列固定步骤, 并应用一般 DNA 荧光染色 DAPI (4 ‘, 6-二胺-2-苯丁胺, 一种 dna 结合荧光染料) 结合到雌性和雄性内核。在这里, 我们提出了技术, 并介绍了它的应用的一个例子, 以测试是否有一个内共生细菌, Rickettsia sp. nr。对塔巴奇的主要性别比影响. 这种方法可以帮助白蝇的人口研究, 或确定是否存在性别分配与某些环境刺激。
Introduction
研究性别分配, 或对男性和女性后代的相对投资, 是行为生态学1、2、3 的基石。除了测试行为适应性模型的能力外, 了解一个生物的性别分配策略还可以改进其种群动态模型。在许多物种中, 性别分配由母亲控制。为了确定性别分配, 重要的是要确定主要的性别比率或女性在卵子沉积时的比例。虽然成人出现的性别比率可能为性别分配提供线索, 但男女青少年之间不同的发育死亡率通常可能会大大扭曲成人性别比率。在一些物种的膜翅目动物, 包含蚂蚁, 蜜蜂和黄蜂的昆虫的顺序, 主要的性别比已确定与细胞遗传学检测, 染色的胚胎, 以查看基因 DNA。由于膜翅目是单倍体, 新生雄蛋是单倍体, 仅含有雌性前核 (n), 而新生的雌卵是二倍体, 同时含有雄性和雌性的前粒 (2n)。尽管被称为白翅目的真正虫子 (半翅目) 的萨比生类动物阿莱罗迪达伊也是单倍体, 但目前还没有确定的方法来寻找这些昆虫的主要性别比。考虑到对这个家庭中为数不多的世界性严重害虫的研究强度, 以及性别比例在白蝇4、5、6、7的竞争互动中的重要性, 这或许令人惊讶 ,8,9, 10 和一般的人口动态。在单倍体昆虫中, 性别比率也不受性别测定系统的限制, 从而有可能进行选择性受精, 并使不同环境而不稳定的性别比率。在这里, 我们提出了一种技术, 以确定主要性别比例的物种复合体的白蝇统称为甘薯白蝇, b. 塔巴奇。这个物种名称包括全球28种以上的 11种物种, 其中包括一些最具破坏性的全球入侵害虫 12,13.应用这种技术来确定 b . tabaci和其他 Aleyrodidae 中的性别分配模式, 将允许对变量进行更严格的调查, 包括温度、寄主植物、内共生细菌或平面/白粉可能会影响白度主要性别比率和白度种群动态。
我们不知道任何类似的鸡蛋染色技术 b . 塔巴奇。与其他昆虫卵14使用的染色方法相比, 该方案非常方便, 因为它省略了隔夜固定步骤, 因此可以在3小时内完成。作为一个应用的例子, 一种内共生细菌, Rickettsia sp . nr。bellii,与女性偏见有关, 在我们的实验室线b. tabaci中东-亚细亚小鱼 (meam1)15,16。在 b . tabaci meam1 实验室线路 (“TABACI”, 从 Maricopa 农业中心收集) 中, 我们测试受 Rickettsia(r+) 感染的雌性是否比未感染 (r-) 雌性给更多的卵受精。
Protocol
Representative Results
Discussion
该方案首次记录了塔巴奇的受精率或主要性别比率。这个协议的挑战是, 它需要研究人员学习如何快速处理白卵, 确保鸡蛋经过不超过 1小时, 直到它们被固定。在初步实验中, 在3小时或更长时间的产卵期固定的卵太旧, 无法观察受精, 因为已经发生了同步, 有丝分裂正在进行中。在1到3小时之间, 原核呈圆形。虽然两个细胞核的早期存在表明受精卵, 但稍晚一点, 两个细胞核在准备合成合子时的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究的资金来自国家安全基金向 m. s. h. 提供的赠款 (deb-1020460) 和美国农业部 AFRI 赠款 (2010-03752)。作者感谢布伦南·泽尔染色白蛋与许多技巧和禅宗。作者感谢迈克·瑞勒允许使用他的荧光显微镜进行成像。作者感谢苏珊娜·凯利和马可·格比奥拉的蛋象。作者感谢苏珊娜·凯利和吉米·康威在实验的关键时刻提供的帮助。
Materials
| 1XPBS | Any | ||
| 1XTBST | Any | 5X solution made from 30g Tris, 43.8g NaCl, 5 mL Tween-20 and 1.0g NaN3 pH7.5, and brought to 1L with PCR grade water | |
| Bleach | Clorox | Any household bleach will work as long as it can be diluted to 0.83% Sodium hypochlorite | |
| Clear nail polish | Any | ||
| DAPI dilactate | Santa Cruz Biotechnology | sc300415 | |
| Ethanol | Any | Dilute to 70% EtOH | |
| Fluorescent microscope | Nikon | Nikon Eclipse 50i was used in this experiment, but any fluorescent microscope with 340/380 nm excitation filter and at least 4-10X magnification can be used | |
| Glacial acetic acid | Mallinckrodt | UN2789 | |
| Glycerol | Any | ||
| Microscope | Wild | A Wild M5A microscope was used for this experiment, but any microscope where the operator can clearly see the whitefly eggs can be used | |
| Microscope slide covers | Any | Methods are for 18mm x 18mm sized slide covers. More mounting media will need to be added for larger slide covers. | |
| Microscope slides | Any | ||
| Minuten nadel pins | BioQuip | 1208SA | Minuten nadel pins are optional for fashioning as probes with pipette tips |
| NaCl | Any | ||
| NaN3 | Any | ||
| n-propyl-gallate | Sigma/Santa Cruz Biotechnology | P3130/sc-250794 | |
| Parafilm | Bemis | ||
| Pasteur pipettes | Fisher Scientific | 13-678-20A | Fisherbrand Disposable Borosilicate glass Pasteur pipettes 5.75 in. A Bunsen burner may also be needed if operator would like to lengthen and narrow pipettes |
| PCR grade water | Any | ||
| Pipette tips | Any | Pipette tips are optional for fashioning as probes with minuten nadel pins | |
| Small dropper bulb | Any | Must fit on Pasteur pipette | |
| Tris | Any | ||
| Tween-20 | Any |
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