Name: improved fecundity and fertility assay for aedes aegypti using 24 well tissue culture plates (eagal plates)
Uploaded: 2021-05-04T12:30:00
Description: Watch this Scientific Journal Video about Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates at JoVE.com

我们感谢德克萨斯州农业研究昆虫病媒疾病赠款计划的资金。我们还感谢阿德尔曼实验室成员在起草手稿时帮助开发这种方法和建议，以及凯文·迈尔斯实验室成员。我们还感谢评论者和编辑们的帮助，使他们的手稿更好。

1. 板准备
<ol><li>使用约 1.6 毫米（1/16 在）位（图 2）的家用钻孔在 24 个井组织培养板盖（每口井 4 −6 孔）中钻孔。 注意：这些孔防止水凝结从糖脂积聚在盖子上，蚊子可能会在那里产卵。雌性 Ae.aegypti（" 利物浦"菌株）的标准尺寸为~3.11毫米翼展。建议使用较小的蚊子避免从盘子中逃出时缩小孔的大小。</li>
	<li>在渗漏实验前一天，在室温下彻底清洗和冲洗盘子?...

<ol>
	<li>Bond, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+irradiation+dose+to+Aedes+aegypti+and+Ae.+albopictus+in+a+sterile+insect+technique+program.">Optimization of irradiation dose to Aedes aegypti and Ae. albopictus in a sterile insect technique program.</a> PLoS One. 14 (2), 0212520 (2019).</li><li>Fernandes, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+aegypti+larvae+treated+with+spinosad+produce+adults+with+damaged+midgut+and+reduced+fecundity.">Aedes aegypti larvae treated with spinosad produce adults with damaged midgut and reduced fecundity.</a> Chemosphere. 221, 464-470 (2019).</li><li>Inocente, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticidal+and+Antifeedant+Activities+of+Malagasy+Medicinal+Plant+(Cinnamosma+sp.)+Extracts+and+Drimane-Type+Sesquiterpenes+against+Aedes+aegypti+Mosquitoes.">Insecticidal and Antifeedant Activities of Malagasy Medicinal Plant (Cinnamosma sp.) Extracts and Drimane-Type Sesquiterpenes against Aedes aegypti Mosquitoes.</a> Insects. 10 (11), 373 (2019).</li><li>Marrelli, M. T., Moreira, C. K., Kelly, D., Alphey, L., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+transgenesis:+what+is+the+fitness+cost.">Mosquito transgenesis: what is the fitness cost.</a> Trends in Parasitology. 22 (5), 197-202 (2006).</li><li>da Silva Costa, G., Rodrigues, M. M. S., Silva, A. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+blood-free+diet+for+Anopheles+darlingi+(Diptera:+Culicidae).">Toward a blood-free diet for Anopheles darlingi (Diptera: Culicidae).</a> Journal of Medical Entomology. , 217 (2019).</li><li>Gonzales, K. K., Tsujimoto, H., Hansen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+serum+and+BSA,+but+neither+red+blood+cells+nor+hemoglobin+can+support+vitellogenesis+and+egg+production+in+the+dengue+vector+Aedes+aegypti.">Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti.</a> PeerJ. 3, 938 (2015).</li><li>Gonzales, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+SkitoSnack,+an+Artificial+Blood+Meal+Replacement,+on+Aedes+aegypti+Life+History+Traits+and+Gut+Microbiota.">The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota.</a> Scientific Reports. 8 (1), 11023 (2018).</li><li>Tsujimoto, H., Anderson, M. A. E., Myles, K. M., Adelman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Candidate+Iron+Transporters+From+the+ZIP/ZnT+Gene+Families+in+the+Mosquito+Aedes+aegypti.">Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti.</a> Frontiers in Physiology. 9, 380 (2018).</li><li>Ioshino, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviplate:+A+Convenient+and+Space-Saving+Method+to+Perform+Individual+Oviposition+Assays+in+Aedes+aegypti.">Oviplate: A Convenient and Space-Saving Method to Perform Individual Oviposition Assays in Aedes aegypti.</a> Insects. 9 (3), 103 (2018).</li><li>Price, D. P., Schilkey, F. D., Ulanov, A., Hansen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+mosquitoes,+large+implications:+crowding+and+starvation+affects+gene+expression+and+nutrient+accumulation+in+Aedes+aegypti.">Small mosquitoes, large implications: crowding and starvation affects gene expression and nutrient accumulation in Aedes aegypti.</a> Parasites &amp; Vectors. 8, 252 (2015).</li><li>Valerio, L., Matilda Collins, C., Lees, R. S., Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+vector+arthropod+culture:+an+example+using+the+African+malaria+mosquito,+Anopheles+gambiae+(Diptera:+Culicidae).">Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae).</a> Malaria Journal. 15 (1), 262 (2016).</li><li>Rueden, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529 (2017).</li></ol>

与英国《金融时报》的方法相比，EAgaL板块大幅减少了在伊蚊进行个体生育和生育检测的劳动、时间和空间。FT 方法与 EAgaL 板之间的初步比较导致所有步骤的使用时间缩短（成像技术应用于 FT 方法）（表 1）。作为参考，在表 2中提供了启动和每次检测（一个 24 井 EAgaL 板与 24 个 FTs）成本的估计值。
使用 EAgaL 板时需要考虑一些问题。最...

控制蚊子以保护人类免受病媒传播病原体的伤害是一项重要的公共卫生目标，主要是因为缺乏对蚊子携带的大多数病原体的有效疫苗。许多研究旨在减少蚊子的适应与一个领域适用的人口减少策略1，2，3。这包括广泛的研究，以创建转基因蚊子和/或CRISPR/Cas9淘汰赛线。这种人口调整方法需要详细评估个别的健身参数4。评估雌性蚊子健康状况的传统实验室技术包括：在100毫升容器5中单独密封配对、供血喂养的雌性蚊子，修改50毫升圆锥管，或使用湿棉和过滤纸盘（即蛋纸）1、2、6、7进行湿润表面改造的果蝇饲养管。这种方法需要相对较大的空间（例如，30厘米×30厘米x10厘米：W×L x H，最多100个果蝇管）（图1），以及操作单个蛋纸来计算卵子和孵化幼虫，这可能是劳动密集型的。这份手稿提出了一种计算蚊子卵子的方法，并用24个油井板确定孵化率，并作为卵子表面来规避这些问题。
同时，Ioshino等人9 描述了一种使用12个和24个井板进行从女性个体获得的卵子计数的详细方法。他们的协议代表了在节省时间和空间9的传统方法的重大改进。然而，他们描述的协议继续使用湿滤纸作为渗透的表面，这需要展开每个单个纸张来获得计数，因为鸡蛋经常在下面或褶皱中发现。他们的协议也不包括使用成像技术或幼虫计数的方法。
呈现的是一种改进的方法，用于执行卵号（即丰度）和孵化率（即生育率）的健身检测，使用 agarose 作为 24 井组织培养板格式的卵剂表面，用于 Ae. aegypti， 在潮湿表面上渗出。这些板块被命名为"EAgaL"板块，来自 Egg、 阿加玫瑰和 Larva。这24个井板为单个蚊子提供最小的表面产卵，从而简化和大大减少了计算和维持卵子和孵化幼虫几天所需的时间和精力。EAgaL板使用半透明的糖脂作为卵泡表面，无需处理蛋纸，并在孵化时找到卵子和幼虫：拍摄每口井都建立了结果的长期存档记录，并将时间和空间中的计数过程与时间通常有限的饲养/处理过程区分开来。

<table>
	<tbody>
		<tr>
			<td>1.6 mm &#934; drill bit</td>
			<td></td>
			<td></td>
			<td>alternatively heated nails can be used</td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips (long)</td>
			<td>Olympus plastics</td>
			<td>24-165RL</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Thermo Scientific</td>
			<td>930186</td>
			<td>clear, flat-bottom with ringed lid plates</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>VWR</td>
			<td>0710-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact digital camera</td>
			<td>Olympus</td>
			<td>TG-5/TG-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer (Windows, Mac or Linux)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (imageJ) software</td>
			<td></td>
			<td></td>
			<td>download from: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>sharp forceps may break mosquito&#39;s body</td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household bleach</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household electric drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>illuminator for stereomicroscope (gooseneck)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-1000 pipette</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paint brushes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td></td>
			<td></td>
			<td>to record digital camera images (DSHC, SDXC should be better)</td>
		</tr>
		<tr>
			<td>Spreadsheet software (Microsoft Excel)</td>
			<td>Microsoft</td>
			<td></td>
			<td>Any spreadsheet software works</td>
		</tr>
		<tr>
			<td>TetraMin fish food</td>
			<td>Tetra</td>
			<td></td>
			<td>ground with coffee grinder, blender or morter &#38; pestle</td>
		</tr>
		<tr>
			<td>Transfer pipetts</td>
			<td>VWR</td>
			<td>16011-188</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved fecundity and fertility assay for aedes aegypti using 24 well tissue culture plates (eagal plates)

蚊子作为各种病原体的媒介，是一个严重的公共卫生问题。对于那些需要评估蚊子适应参数的研究，特别是鸡蛋的产生和个别水平的孵化率，由于高劳动强度和实验室空间要求，传统方法给调查人员带来了沉重的负担。描述是一个简单的方法，使用24个井组织培养板与每个井的糖和数字成像每个井，以确定鸡蛋数量和孵化率在个别水平，大大减少了时间和空间的要求。

蚊子被注射dsRNA针对候选铁运输机（FeT）或控制基因（EGFP），血液喂养，并测量的丰度和生育能力输出使用EAgaL板法，按照上述程序。
在 dsRNA 注射后，FeT 表达沉默的蚊子的卵子数量和孵化率均显著降低（图 11A−C）。所有控制和治疗蚊子都放置在EAgaL板后72小时PBM。沉默的蚊子也表现出延迟排泄和小而浅色的鸡蛋（图12A，B）?...

Watch this Scientific Journal Video about Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates at JoVE.com

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

描述的是一种节省时间和空间的方法，使用24个井组织培养板来计算卵子和确定单个蚊子的孵化率，从而大大提高肥大和生育检测的规模和速度。

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

这个程序使蚊子研究人员能够在个人层面上轻松地解决蚊子的生殖健康问题。与传统方法相比，该协议显著减少了在个人层面测试蚊子的生育能力和生育能力所需的时间、空间和人工。首先为渗漏实验准备盘子。使用 1.6 毫米位的家用钻头，在 24 井培养板的盖子上每口井钻 4 到 6 个孔。实验前一天，在室温下清洗和冲洗盘子，将盘子浸泡在1%至5%的漂白剂中，浸泡30至60分钟。然后，在运行中的除离子水中彻底冲洗板并干燥。将2%的蔗糖融化在除离子水中，并立即在24井板的每口井中加入500微升的熔融糖。将盘子放在实验室的长椅上过夜，以干燥任何凝结物。在喂血前至少16小时，从雌性蚊子身上去除任何水源或糖。喂食当天，将水循环器加热至37摄氏度，用放置在人工喂食器中的脊椎动物血液喂养蚊子15至30分钟。然后，将蚊子转移到冰上的玻璃盘上。选择那些充满血液的容器，并把它们放入一个有30%蔗糖水的容器中。女性至少需要72小时才能完成排泄和卵子发育。在将蚊子转移到24井板前约一小时，使用转移移液器将两到三滴水加入每口井中。用二氧化碳击倒蚊子，并把它们转移到冰上的玻璃盘上。然后，将每只蚊子单独放在24井板的倒盖上。一旦所有24只蚊子被放置，用倒板底部盖住盖子，用新的橡皮筋固定盖住盖子，并将其放置在环境室中，直到蚊子恢复。然后，将盘子右侧向上转动。允许雌性蚊子在24至48小时内渗出。然后，通过将雌性从盘子中释放到一个大笼子里来取出它们。在计算鸡蛋之前，检查每口井的鸡蛋在井壁上，在糖和塑料表面的边缘，他们很难在照片中解决。使用湿画笔将这些鸡蛋移到平坦的表面，使所有鸡蛋均匀，并且不会相互重叠。使用钳子去除井中可能干扰成像卵的任何断腿、翅膀和其他颗粒。在显微镜模式下设置紧凑型数码相机，使用户能够聚焦在近一厘米的物体上，并拍摄每口井的图像。拍摄每口盘子的油井后，拍摄整个盘子的图像。稍后使用成像订单标签来区分每个板。在每口井中加入大约五滴水，以防止其糖塞和卵子干燥，并诱导胚胎发育和孵化。将图像传输到计算机，用板和井 ID 重命名文件，以便于组织。打开图像与图像J，并使用多点工具标记每个鸡蛋，放大或缩小，以计算鸡蛋团。标记所有鸡蛋后，双击多点图标以增加标记数并将结果记录在电子表格中。一旦孵化出的幼虫出现，就开始向井中添加食物。大约五到八天后，用碎冰覆盖板15至20分钟，对幼虫进行麻醉。在板下插入黑色材料，以增强对比度，并在保持板在冰上的同时拍摄每口油井的图像。拍摄完每口油井后，用成像顺序标签拍摄整个盘子的图像。打开图像J，并使用多点工具来计算幼虫。幼虫在形状、角度和焦点上可能有所不同。不包括棚角质层，看起来像只有头部的幼虫与一点点的身体，或缩小的幼虫。在电子表格中记录结果。蚊子被注射双链RNA瞄准候选铁运输机，FeT，或控制基因EGFP。然后，他们用鹰板法进行血液喂养和生育能力输出测量。沉默的铁运输机表达的蚊子在卵子数量和孵化率上都显著下降。沉默的蚊子也表现出延迟排泄物和小而浅色的鸡蛋。拍摄好照片是这种方法的关键一步，因为单个卵子或幼虫的图片质量和数量直接影响结果的质量。

Research

JoVE Journal

Biology

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

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科研

生物学

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It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

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RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

准备辐照和标记的雄性 埃及伊蚊 ，以便在操作性昆虫技术计划中释放

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

使用 24 个井组织培养板（EAgaL 板）改进了伊蚊的丰度和生育率测定