睾丸融合的显微外科梗阻
Summary
将铝箔显微外科插入 Spodoptera litura 的睾丸之间，以阻碍睾丸的融合。该过程包括冷冻，固定，消毒，切口，放置屏障，缝合，术后喂养和检查。这种方法提供了一种干扰组织形成的方法。
Abstract
没有使用RNA干扰（RNAi）和簇状规则间隔的短回文重复序列（CRISPR）/ CRISPR相关的核酸内切酶Cas9等遗传方法，而是在 Spodoptera litura 的睾丸之间插入物理屏障，以研究这种显微手术对其生长和繁殖的影响。在睾丸之间插入铝箔后，昆虫在变质过程中的蜕皮正常进行。昆虫的生长和发育没有显着改变;然而，如果显微外科手术停止睾丸融合，精子束的数量就会发生变化。这些发现表明，阻断睾丸融合会影响男性生殖能力。该方法可进一步应用于中断器官之间的通信，以研究特定信号通路的功能。与传统手术相比，显微外科手术只需要冷冻麻醉，比二氧化碳麻醉更可取。显微外科手术还可以最大限度地减少手术部位区域并促进伤口愈合。但是，具有特定功能的材料的选择需要进一步研究。在手术过程中做切口时，避免组织损伤至关重要。
Introduction
融合是组织或器官发育中的常见现象。例子包括 果蝇1中的背侧闭合和胸部闭合以及小鼠和鸡的腭形态发生，神经管形态发生以及心脏形态发生2。CRISPR和RNAi已被应用于研究基因在融合过程中的作用2，3，4。
Spodoptera litura（S. litura，鳞翅目： Noctuidae）是一种有害的多食性害虫，广泛分布在亚洲的热带和亚热带地区，包括中国4，5，6。 S. litura 的广泛分布部分归因于其强大的生殖能力，这与性腺发育有关。男性不育症是控制这种害虫的一种方法。如睾丸结构示意图所示，睾丸被睾丸鞘包围，包括外鞘（腹膜鞘）和内基底层。基底层向内延伸以形成滤泡上皮，并将睾丸的内部区域分成四个称为卵泡的腔室（图1）。
在卵泡中，精子形成有丝分裂和减数分裂后发育成精子，然后精子囊中的精子朝同一方向排列形成精子束7。在精子发生过程中，初级精子细胞分化为真芘精子或芘精子。幼虫期的精子细胞发育成真芘精子，长尾巴连接到细长的细胞核的头部;这些可以使卵子受精。相反，蛹中期的精子细胞发育成具有废弃细胞核的芘精子;这些精子有助于芘啉精子的存活、运动和受精9，10。蛹的第 6天是睾丸具有丰富的真芘和芘精子束的时期。
图 1：鳞翅目昆虫睾丸结构示意图11。请单击此处查看此图的放大版本。
睾丸融合发生在鳞翅目的大多数昆虫11，12中，特别是在那些农业害虫中。睾丸融合是指一对睾丸在幼虫期双侧生长，彼此接近并粘附，最终整合成单个性腺11。在 Spodoptera litura 中 ，它发生在从幼虫到蛹阶段的过程中。从5龄（L5D1）的第 1天到6龄（L6D4）的第 4天，这对睾丸的大小逐渐增长，颜色从象牙白色变为浅黄色。当它到达前期（L6D5至L6D6）时，它会变成淡红色。两个双侧对称睾丸在前裆阶段相互接近，融合为一个，逆时针扭曲（多拉视图），在蛹期和成虫期产生单个睾丸11。这种现象不会发生在蚕身上，蚕具有相当大的经济重要性，并且已经驯化了5000年13。因此，假设睾丸的融合提高了生殖能力。
为了确定 踞 踤荠睾融合术的意义，重要的是要研究阻断该过程的影响。在该协议中，在睾丸之间微外科插入铝箔以保持它们分离，并研究了昆虫及其睾丸发育的相应变化。
Protocol
Representative Results
Discussion
在 对Spodoptera litura的睾丸融合进行显微外科手术后，精子束的数量减少，这支持了这种融合有利于生殖能力的假设。自20世纪初 以来，外科手术一直被用于研究昆虫的生理发育。为了确定颅神经是否受到昆虫的调节，一些研究人员对不同的昆虫（包括半翅目 Rhodnius prolixus ，鳞翅目中的 Lymantria dispar ）进行了结扎和斩首等程序 15 ， <sup class="xre…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（Nos.：31772519，31720103916;）和西南大学蚕基因组生物学国家重点实验室（编号：sklsgb2013003）的公开资助。
Materials
| 75% Rubbing alcohol | Qingdao Hainuo Nuowei Disinfection Technology Co., Ltd | Q/370285HNW 001-2019 | |
| Colored Push Pins | Deli Group Co.,LTD | 0042 | |
| Corneal Scissors | Suzhou Xiehe Medical Device Co., Ltd | MR-S221A | Curved and blunt tip |
| Glad Aluminum Foil | Clorox China(Guangzhou) Limited | 831457 | 10 cm*2.5 cm*0.6 |
| Medical Cotton Swabs (Sterile) | Winner Medical Co., Ltd. | 601-022921-01 | |
| Medical Iodine Cotton Swab | Winner Medical Co., Ltd. | 608-000247 | |
| Needle holder | Shanghai Medical Instruments (Group) Ltd., Corp. | J32030 | 14 cm fine needle |
| Sterile surgical blade | Shanghai Pudong Jinhuan Medical Supplies Co., LTD | #11 | |
| Suigical Blade Holder | Shanghai Pudong Jinhuan Medical Supplies Co., LTD | K6-10 | Straight 3# |
| Suture thread with needle | Ningbo Medical Stitch Needle Co., Ltd | needle: 3/8 Circle, 2.5*8 ; Thread: Nylon, 6/0, 25 cm | |
| Tying Forceps | Suzhou Xiehe Medical Device Co., Ltd | MR-F201T-3 | Straight-pointed; long handle; 0.12 mm-wide-head |
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