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Nictation is a dauer-specific standing and waving behavior of the nematodes including Caenorhabditis species. Nictation enhances the capability of free-living nematodes to hitchhike to other animals as well as parasitic nematodes to infect their hosts. However, lack of an assay for this behavior has made it difficult to elucidate its underlying regulatory mechanisms and related genetic pathways. We have developed nictation assays that enable the quantification of the nictation behavior of individuals and groups of worms. Gauze assay is less quantitative but is an easier way to observe nictation behavior in plates with plenty of dauers. The micro-dirt chip made from PDMS mold is a more sophisticated method to quantify the nictation behavior. Nictation can be quantified on a micro-dirt chip either by measuring the average nictating time of individual dauers or by the fraction of nictating worms in a given dauer population.

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Nictation Assays for Caenorhabditis and Other Nematodes
杆状线虫和其它线虫的Nictation行为分析

神经科学 > 行为神经科学 > 线虫行为
作者: Daehan Lee
Daehan LeeAffiliation: Baisc Research Center, School of Biological Sciences, Seoul National University, Seoul, Korea
Bio-protocol author page: a2089
Harksun Lee
Harksun LeeAffiliation: Baisc Research Center, School of Biological Sciences, Seoul National University, Seoul, Korea
Bio-protocol author page: a2090
Myung-kyu Choi
Myung-kyu ChoiAffiliation: Baisc Research Center, School of Biological Sciences, Seoul National University, Seoul, Korea
Bio-protocol author page: a2091
Sungsu Park
Sungsu ParkAffiliation: School of Mechanical Engineering & Department of Global Biomedical Engineering, Sungkyunkwan University, Suwon, Korea
Bio-protocol author page: a2092
 and Junho Lee
Junho LeeAffiliation 1: Baisc Research Center, School of Biological Sciences, Seoul National University, Seoul, Korea
Affiliation 2: Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea
Affiliation 3: Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea
For correspondence: elegans@snu.ac.kr
Bio-protocol author page: a2093
Vol 5, Iss 7, 4/5/2015, 2629 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1433

[Abstract] Nictation is a dauer-specific standing and waving behavior of the nematodes including Caenorhabditis species. Nictation enhances the capability of free-living nematodes to hitchhike to other animals as well as parasitic nematodes to infect their hosts. However, lack of an assay for this behavior has made it difficult to elucidate its underlying regulatory mechanisms and related genetic pathways. We have developed nictation assays that enable the quantification of the nictation behavior of individuals and groups of worms. Gauze assay is less quantitative but is an easier way to observe nictation behavior in plates with plenty of dauers. The micro-dirt chip made from PDMS mold is a more sophisticated method to quantify the nictation behavior. Nictation can be quantified on a micro-dirt chip either by measuring the average nictating time of individual dauers or by the fraction of nictating worms in a given dauer population.
Keywords: nictation(nictation 行为), c. elegans(秀丽隐杆线虫), dauer(休眠), micro-dirt chip(微污垢芯片), gauze assay(纱布测定)

[Abstract] 捕食是线虫(包括Caenorhabditis)物种的持久性和持续性行为。 捕食提高了自由生活线虫与其他动物以及寄生线虫感染其宿主的能力。 然而,缺乏这种行为的测定使得难以阐明其基础调节机制和相关的遗传途径。 我们已经开发了定义测定,使得能够量化的个人和蠕虫组的nictation行为。 纱布测定较少定量,但是是更容易的方法来观察具有大量持久的板的行为。 由PDMS模具制成的微尘片是一种更复杂的方法来量化叙述行为。 可以通过测量个体动物的平均哺乳时间或通过在给定动物群体中的ating虫的分数,在微尘片上量化捕食。

Materials and Reagents

  1. Dauer larva
  2. SU-8 photoresist 2008 (MicroChem Corp)
  3. Polydimethylsiolaxne (PDMS) (Sylgard® 184 Silicone Elastomer Kit, Dow-Corning)
  4. Agar powder (OCI Company Ltd, catalog number: 9002-18-0 )
  5. Cotton medical gauze
  6. M9 solution (see Recipes)
  7. Synthetic pheromone plates (see Recipes)

Equipment

  1. Microwave oven or autoclave
  2. Dissecting microscope (Leica Microsystems, model: S6E )
  3. Mouth pipette with glass capillary (Chase)
  4. 60Φ Petri dish (SPL Life Sciences, model: 10060 )
  5. Slide glass (Marienfeld)
  6. Spatula
  7. Glass flask
  8. Incubator (Vision Scientific Co. Ltd., model: VS-8480S )

Procedure

  1. Prepare dauers for nictation assay
    1. Dauers can be induced by various methods. Most easily, dauer can be prepared from starved plates. Dauer can also be induced by treating crude pheromone (Golden and Riddle, 1984). We usually use synthetic pheromone plates to induce dauers, and those dauers seem to give more reproducible data than starvation-induced dauers. Dauers of temperature-sensitive dauer-formation constitutive mutants can be easily induced by temperature shift. We usually let adult worms to lay eggs in 15 °C and then shift temperature to 25 °C.

  2. Gauze assay
    1. Count total number of dauers in the plate to assay.
    2. Cut cotton medical gauze to fit to the size of petri dish, and put it on. Dauers waving on filament would be observed within 5 min.
    3. Count the number of dauers waving or standing on gauze at a moment 3 times. Nictation ratio can be measured as [Average number of nictating dauers/Number of total dauers].

      Video 1. Gauze assay

      To play the video, you need to install a newer version of Adobe Flash Player.

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  3. Micro-dirt chip manufacture
    1. A micro-dirt master was fabricated by patterning an array of holes [25 μm (radius) x 25 μm (height) x 25 μm (distance)] onto a 4-inch silicon wafer by photolithography with SU-8. Negative replicas of the master were made by pouring 10 ml of PDMS prepolymer onto the master and curing the polymer at 65 °C for 30 min (Xia and Whitesides, 1998). The cured polymeric layer was peeled off from the master and cut to fit in 60Φ petri dish. PDMS negative replicas function as a mold for micro-dirt chip. Make agar solution (3.5%~4%) with D.W. by boiling with microwave or autoclave. Cool it to about 70 °C, and pour agar solution onto the PDMS mold placed in a 60Φ petri dish with structure-side up.
    2. Remove air bubbles on the surface of PDMS mold with slide glass before agar solution is solidified. Optimize agar concentration considering quality of agar powder and stiffness of agar solution. If solution is too stiff, it hinders air bubble removal. If air bubbles are removed, wait enough until agar solution is fully solidified.
    3. Using spatula, take out the solidified part (PDMS mold + micro-dirt chip) from petri dish, and detach PDMS mold from it. Remaining agar part is micro-dirt chip with positive array of posts.
    4. Dry micro-dirt chip in 37 °C incubator (without shaking) for 1.5 h before use.

  4. Micro-dirt chip assay
    1. Collect 30+ dauers with M9 loaded mouth pipette, and release them on the center of micro-dirt chip under dissecting microscope. It takes 10~30 min for dauers to start locomotion after transfer to micro-dirt chip in room temperature.
    2. Only dauers can nictate on micro-dirt chip. When dauer lift their neck, we regard the point as nictation initiation. When its body lands back to surface, we regard it as completion of nictation.
    3. For individual nictation assay, observe each moving dauer for 1 min and measure nictating time. Quiescent dauers (including standing dauers) are excluded from test. From these raw data, nictation ratio, initiation index, and average duration can be extracted according to Lee et al. (2011).
    4. For population nictation assay, scan micro-dirt chip (by eye) while counting number of nictating dauers and crawling dauers (Figure 1). Only moving (crawling and nictating) dauers should be counted. Initiation index and average duration cannot be extracted from population assay.


      Figure 1. Micro-dirt chip population nictation assay. The surface of micro-dirt chip is scanned by eye to count the number of moving dauers and nictating dauers. Quiescent dauers are excluded. Nictation ratio can be measured as fraction of nictating dauers among moving dauers. We measure 3 times for same micro-dirt chip and average the result. For each strain, we usually make 5+ independent replicates to get reproducible nictation ratio.

      Video 2. Micro-dirt chip manufacture and assay

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

Notes

  1. Nictation assay results exhibit considerable day to day variation, and the cause of stochastic results is unknown. To overcome the problem of variability and reproducibility, it is recommended to make multiple independent replicates rather than to test many worms at once. For instance, N2 might exhibit nictation ratio from 10% to 70% from one test (N>=10) by individual assay, but one will get stable average nictation ratio about 20%~40% from multiple tests.
  2. In nictation assay, we only measure moving dauers. For individual nictation assay, a waving worm can suddenly stand quiescently, then we exclude that worm from the result. However, we also count nictating worms which might stop after a moment in population assay. Presumably, this difference makes population assay to yield slightly higher nictation ratio than individual assay.

Recipes

  1. M9 buffer
    3 g KH2PO4
    6 g Na2HPO4
    5 g NaCl
    1 ml 1 M MgSO4
    H2O to 1 L
    Sterilized by autoclaving
  2. Synthetic pheromone plate
    1.5 g KH2PO4
    0.25 g K2HPO4
    1 g NaCl
    10 g agar
    (or alternatively, 5 g agar + 3.5 g agarose for digging strains like HW or npr-1 mutant)
    4 mg cholesterol (add after autoclaving)

Acknowledgments

This work was supported by research grants through the National Research Foundation of Korea (NRF) (NRF-2014R1A4A1005259, NRF-2013K2A3A1000102 and NRF-2013R1A2A1A03070982). D. L. was supported by TJ Park Science Fellowship from the POSCO TJ Park Foundation. This protocol has been adapted and modified from our previous work, Lee et al. (2012).

References

  1. Golden, J. W. and Riddle, D. L. (1984). A Caenorhabditis elegans dauer-inducing pheromone and an antagonistic component of the food supply. J Chem Ecol 10, 1265–1280.
  2. Lee, H., Choi, M. K., Lee, D., Kim, H. S., Hwang, H., Kim, H., Park, S., Paik, Y. K. and Lee, J. (2012). Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons. Nat Neurosci 15(1): 107-112.
  3. Xia, Y. and Whitesides, G. M. (1998). Soft lithography. Angew Chem Int Edn Engl 28(1): 153-184.

材料和试剂

  1. Dauer幼虫
  2. SU-8光致抗蚀剂2008(MicroChem Corp)
  3. 聚二甲基硅氧烷(PDMS)(Sylgard 184 Silicone Elastomer Kit,Dow-Corning)
  4. 琼脂粉(OCI Company Ltd,目录号:9002-18-0)
  5. 棉纱布
  6. M9解决方案(参见配方)
  7. 合成信息素板(见配方)

设备

  1. 微波炉或高压锅
  2. 解剖显微镜(Leica Microsystems,型号:S6E)
  3. 带玻璃毛细管(Chase)的嘴移液器
  4. 60Φ培养皿(SPL Life Sciences,型号:10060)
  5. 幻灯片(Marienfeld)
  6. 小铲
  7. 玻璃瓶
  8. 孵育器(Vision Scientific Co.Ltd。,型号:VS-8480S)

程序

  1. 准备用于监听测试的dauers
    1. 可以通过多种方法诱导Dauer。 最容易,dauer可以 从饥饿板制备。 也可以通过治疗诱导多柔比星 粗信息素(Golden and Riddle,1984)。 我们通常使用合成 信息素板诱导dauers,那些dauers似乎给了更多 可重复的数据比饥饿诱导的dauers。 Dauers 温度敏感的dauer形成组成型突变体可以很容易 温度漂移引起的。 我们通常让成虫蠕虫产卵 15℃,然后将温度变为25℃

  2. 纱布测试
    1. 计算待测板中的总持续时间。
    2. 切割棉花 医用纱布适合大小的培养皿,并把它。 Dauers 在5分钟内观察到在灯丝上的波动
    3. 计数 数目的持续挥动或站在纱布的时刻3次。 睡眠比可以测量为[平均睡眠次数 dauers /总持有人数]。

      视频1.纱布测试
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  3. 微尘芯片制造
    1. 通过图案化孔阵列制造微污垢母版 μm(半径)×25μm(高度)×25μm(距离)]到4英寸硅上 晶片通过使用SU-8的光刻法。 主副本的副本 通过将10ml PDMS预聚物倒入母板上并固化来制备 聚合物在65℃下30分钟(Xia和Whitesides,1998)。固化 聚合物层从母板上剥离并切割以适合60Φ 培养皿。 PDMS阴性复制品作为微污垢的模具 芯片。用D.W.制备琼脂溶液(3.5%〜4%)。通过用微波煮沸 或高压釜。冷却至约70℃,并将琼脂溶液倒入 PDMS模具放置在结构侧向上的60Φ培养皿中
    2. 在之前用载玻片除去PDMS模具表面上的气泡 琼脂溶液固化。优化琼脂浓度考虑 琼脂粉的质量和琼脂溶液的刚度。如果解决方案 太硬,阻碍气泡去除。如果除去气泡, 等待直到琼脂溶液完全固化
    3. 使用 刮刀,取出固化部分(PDMS模具+微尘片) 培养皿,并从其分离PDMS模具。剩余的琼脂部分 微污垢芯片与正阵列的柱
    4. 在37℃培养箱(无振荡)中干燥微污垢芯片1.5小时,然后使用。

  4. 微污渍芯片测试
    1. 收集30 + dauers与M9加载口吸管,并释放他们   中心的微污垢芯片在解剖显微镜下。 需要10〜30 min,用于在转移到微尘屑片之后开始运动 室温。
    2. 只有持久的人才能在微尘片上。 当dauer抬起他们的脖子,我们认为点作为开始。 当它的身体落回到表面,我们认为它的完成 nictation。
    3. 对于单独的诊断测定,观察每个移动 dauer 1分钟并测量摄取时间。 休息室(包括 站立的dauers)从测试中排除。 从这些原始数据,nictation 比率,起始指数和平均持续时间 到Lee 等人(2011)。
    4. 对于群体诊断测定,扫描 微污垢芯片(通过眼睛),同时计数数量的嘲笑dauers和 爬行动物(图1)。 只有移动(爬行和捕食) 应计算。 起始索引和平均持续时间不能 从群体测定中提取

      图1.微污垢芯片 微粒子芯片的表面被扫描  眼睛计数移动的dauers和nictation dauers的数量。 不包括休息室。情绪比可以测量为 在移动dauers之间的分数。我们测量3次  相同的微尘片和平均结果。对于每个菌株,我们 通常使5 +独立的重复,以获得可重复的陈述 比率
      视频2.微尘芯片制造和测试
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笔记

  1. 记忆测定结果表现出相当大的日常变化,并且随机结果的原因是未知的。 为了克服可变性和重现性的问题,建议进行多次独立的重复,而不是一次测试多个蠕虫。 例如,通过单独测定,N 1 2可以显示出来自一个测试(N> = 10)的从10%至70%的记忆比,但是可以获得从约20%至40%的稳定的平均记忆比 多重测试。
  2. 在诊断测定中,我们仅测量移动停止。 对于个体诊断测定,一个挥动的蠕虫可以突然静止,然后我们从结果中排除该蠕虫。 然而,我们也计数捕食蠕虫,可能在人口测定中停止一会儿。 推测,这种差异使得群体测定产生比单独测定稍高的记忆率

食谱

  1. M9缓冲区
    3g KH sub 2 PO 4 sub
    6g Na 2 HPO 4
    5克NaCl
    1ml 1M MgSO 4 H sub 2 O到1L
    高压灭菌
    灭菌
  2. 合成信息素板
    1.5g KH 2 PO 4 sub/
    0.25g K 2 HPO 4
    1克NaCl
    10g琼脂
    (或者,5g琼脂+ 3.5g琼脂糖用于挖掘如em 或 npr-1 突变体的菌株) 4 mg胆固醇(高压灭菌后加入)

致谢

这项工作是通过国家研究基金会(NRF)(NRF-2014R1A4A1005259,NRF-2013K2A3A1000102和NRF-2013R1A2A1A03070982)的研究拨款支持。 D. L.由TJ公园科学奖从POSCO TJ公园基金会支持。 该协议已经从我们之前的工作(Lee等人,2012年)修改和修改。

参考文献

  1. Golden,J.W.and Riddle,D.L。(1984)。 Caenorhabditis elegans dauer诱导信息素和食物供应的拮抗成分。 J Chem Ecol 10,1265-1280。
  2. Lee,H.,Choi,M.K.,Lee,D.,Kim,H.S.Hwang,H.,Kim,H.,Park,S.,Paik,Y.K.and Lee, 定义,线虫的线虫的分散行为,受制于 IL2 neurons。 Nat Neurosci 15(1):107-112。
  3. Xia,Y。和Whitesides,G.M。(1998)。 软光刻。 Angew Chem Int Edn Engl 28(1):153-184。
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How to cite this protocol: Lee, D., Lee, H., Choi, M., Park, S. and Lee, J. (2015). Nictation Assays for Caenorhabditis and Other Nematodes. Bio-protocol 5(7): e1433. DOI: 10.21769/BioProtoc.1433; Full Text



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