搜索

Chemotaxis and Jumping Assays in Nematodes
线虫趋化性和跳跃运动试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Nematodes have sensitive olfactory perception, which is used to detect and differentiate many volatile odorants. Some odorants are attractive, others repulsive, and yet others evoke no particular response. Chemotaxis assays can be used to determine the role of certain odors in many different behaviors including foraging, predator avoidance, and mate attraction. In addition to chemotaxis, some species of nematodes in the entomopathogenic genus Steinernema can jump, which is thought to play an important role in host-seeking and dispersal (Dillman and Sternberg, 2012). Jumping and chemotaxis assays have been successfully used to identify odorants that stimulate these behaviors in a variety of nematodes (Bargmann et al., 1993; Campbell and Kaya, 1999; Hallem et al., 2011; Dillman et al., 2012; Castelletto et al., 2014). Here a detailed protocol for chemotaxis and jumping assays is provided based on the growing body of literature.

Keywords: Entomopathogenic(昆虫), Chemotaxis(趋化性), Jumping(跳跃的), Behavior(行为), Nematodes(线虫)

Materials and Reagents

  1. Nematodes
    Note: The Caenorhabditis Genetics Center (CGC) is a great resource for many commonly used nematodes (http://cbs.umn.edu/cgc/home), but not all nematodes are available through this resource. For example, entomopathogenic nematodes (EPNs) are not available through the CGC and either need to be collected from field sampling or requested from another researcher’s lab.
  2. Medical grade certified air mixtures of your chosen percent gas to be tested (e.g. 10% CO2) with the balance being N2 (Praxair, catalog number: 7727-37-9 )
  3. Parafilm (Thermo Fisher Scientific, catalog number: 13-374-10 )
  4. Paraffin oil
  5. Nalgene (Thermo Fisher Scientific, catalog number: 8000-0020 ) FTP 1/8” I.D. tubing
  6. Soda Lime (2-5 mm pellets) (Sigma-Aldrich, catalog number: 72073 )
  7. ½ inch Teflon tape
  8. Laser printer transparency film (e.g. Apollo VCG7060E)
  9. Low-pressure system fittings kit (Bio-Rad Laboratories, catalog number: 7318220 )
  10. Sodium azide (Thermo Fisher Scientific, catalog number: BP9221-500 )
  11. BBL-agar (BD) or Difco-agar
  12. 100 x 15 mm petri dishes (VWR International, catalog number: 25384-088 )
  13. 60 x 15 mm petri dishes (VWR International, catalog number: 25384-090 )
  14. 1.55 mm Whatman 1 filter paper (Fisher Scientific, catalog number: 09-805B )
  15. Blunt Luer-Lok needle (e.g. HAMPTON RESEARCH CORP 22s/2”/3)
  16. Disposable 10 ml Luer-Lok syringe (e.g. BD)
  17. Disposable needle (21 gauge, 1.5 inch) (e.g. BD)
  18. 1/8 inch I.D. (inner diameter) PVC tubing
  19. 50 ml gastight syringes (e.g. HAMPTON RESEARCH CORP, model: 1050 )
  20. 10 ml gastight syringes (e.g. HAMPTON RESEARCH CORP, model: 1010 )
  21. Chemotaxis agar (see Recipes)

Equipment

  1. Dissection stereo microscope (e.g. Leica Microsystems, model: Leica M80 )
  2. Infusion syringe pump (e.g. Harvard Apparatus, model: PHD 22/2000 )
  3. 31 gauge drill bit (3.05 mm) (e.g. Alltrade Tools LLC, model: 480902 rotary tool set )
  4. 56 gauge drill bit (1.18 mm) (e.g. Alltrade Tools LLC, model: 480902 rotary tool set )
  5. Drill or dremmel (e.g. Alltrade Tools LLC, model: 480902 rotary tool set )
  6. Anti-vibration platform (Foam bottom with cardboard platform)

Procedure

  1. Chemotaxis assay procedure

    Preparing for chemotaxis assays
    1. Drilling two 3.05 mm holes in a 10 cm petri dish lid, each hole should be 1 cm from the edge along the diameter, exactly opposite each other. Prepare one lid for each assay that will be done simultaneously, but once prepared, lids can be reused.
    2. Attach flexible PVC tubing (1/8 inch inner diameter) to the top of each hole, with the tubing projecting slightly into the inside of the lid (Figure 1A). The end of the PVC tubing going into the plate lid may be wrapped with layers of parafilm to insure a tight fit. The other end of the tubing will be connected to a 50 ml gastight syringe (Figure 1B). The bottom of the assay plates should have the scoring template attached (Figure 2).
    3. The scoring template can be printed on transparency film and attached to the assay plates using small strips of double-sided tape. Scoring regions are 2 cm diameter circles on each side of the plate along the diameter, with the center of the circle 1 cm from the edge of the plate.
    4. Nematodes can be prepared by first adding 1-2 µl of 20% TritonX-100 in water solution to the bottle in which they are contained. The nematodes can then be quantified by placing five 10 µl drops onto a petri dish lid. The nematodes present can be counted and the density of nematodes in the solution can be calculated. A volume sufficient to obtain the number of nematodes desired can then be placed into an Eppendorf tube and allowed to rest for 2 min or more. The nematodes will sink to the bottom and the excess water can be aspirated. The pellet of worms can then be extracted via pipette.

    Gas chemotaxis assay
    1. Immediately prior to starting the assay, fill the 50 ml gastight syringes according to the experiment. For example, if evaluating the attraction of 10% CO2, one syringe will be filled with 10% CO2 while the other syringe will be filled with the air control. The syringes will be depressed at a rate of 0.5 ml/min using a syringe pump (e.g. PHD 22/2000, Harvard Apparatus) for the duration of the assay.
    2. Place approximately 5-10 µl of worm pellet containing ~50-150 nematodes in the center of the assay plate. These nematodes should be stage-matched or synchronous if possible. Different life stages are known to change in their odor preference (Hallem et al., 2011). Each plate should be undisturbed - on an anti-vibration platform - for the duration of the assay. These assays can be run for variable lengths depending on the specific experiment being performed. One hour seems to be standard but it has been shown that 10, 20, 40, and 60 min can yield the same result in some experiments (Hallem et al., 2011).

    Host chemotaxis assay
    1. Host headspace can be tested by placing a number of hosts inside a 50 ml gastight syringe; the number will vary depending on host size.
    2. The attraction of the host headspace can also be tested without the presence of host-produced CO2 by adding a column of soda lime into the assay setup (Figure 1C-D). A six-inch column of Nalgene (8050-0250) FTP 3/16” ID tubing can be filled with 2-5 mm soda lime pellets (Figure 1C). The column is fitted on one end with fitting 731-8226, which can attach to the tubing that runs to the assay plate lid while on the other end of the column is attached to fitting 731-8223 from a low-pressure fittings kit. That end should fit snugly onto the Luer-Lok end of the 50 ml syringe (Figure 1D). The fittings can be wrapped in Teflon tape to ensure an airtight fit.
    3. At the end of the assay, the number of worms in the scoring regions on each side of the plate should be counted and a chemotaxis index should be calculated as:

      In this example the odorant being tested was CO2 while room air is the control, but the same calculation would be used for other odorants, though the control might be different. Assays should only be scored if ≥3 worms moved into the scoring regions, and trials from assay sessions in which all worms exhibited the same directional bias should be discarded.
    1. The assay as described is meant to be used as a population assay, with ~50-150 nematodes in the experiment but can be used for single nematode experiments.

    Odorant chemotaxis assays
    1. For odorant chemotaxis assays, place 1 μl of 1 M sodium azide in the center of each scoring region as an anesthetic. Place 5 μl of odorant in the center of one scoring region, while placing 5 μl of a control (either paraffin oil, dH2O, or ethanol) in the center of the other scoring region.
    2. As with the gas assays, place ~2 µl of worm pellet containing ~50-150 nematodes in the center of the assay plate. Leave the assay plates undisturbed and score them after either 1 h (for host and CO2 chemotaxis assays) or 3 h (for odorant assays). An alternative assay could be done without the use of sodium azide where assay plates are scored immediately after the allotted experimental time has passed. The odorant assay can also be done without an anesthetic, but then plates would need to be scored immediately after the experiment finishes.


      Figure 1. Chemotaxis assay setup. A. A picture of the 10 cm assay plate lid with two 3.05 mm holes in it. Each hole is 1 cm from the edge along the diameter, exactly opposite each other. The 1/8 inch I.D. PVC tubing is wrapped in parafilm and then fit into the holes. B. A picture of the 1/8 inch tubing attached to a gastight syringe. C. A picture of the soda lime column, constructed using Nalgene (8050-0250) FTP 3/16” I.D. tubing with low-pressure fittings on either end. In the picture, the column is empty but in the assay would be filled with 2-5 mm pellets of soda lime. D. A picture of the soda lime column attached to a gastight syringe and the PVC tubing that connects to the assay plate lid.


      Figure 2. A template used to score chemotaxis assays. Nematodes are placed in the starting location at the beginning of the assay and their attraction to chemical odorants is evaluated as the nematodes distribute in the assay arena.

    Proclivity for Chemotaxis assay
    1. As an extension of the chemotaxis assay, an alternative template can be used to score the nematodes for both the chemotaxis index and a proclivity for chemotaxis index (PCI). The PCI template (Figure 3) can be printed on transparency paper and attached to assay plate with double-sided tape.
    2. During scoring the number of nematodes found in each circle should be recorded as normal. Then, each region should be scored and the number of nematodes in each region should be recorded.
    3. The data can then be used to calculate the percentage of the population in each region.


      Figure 3. A template used to score chemotaxis assays for both chemotaxis index (CI) and proclivity for chemotaxis index. Nematodes are placed in the starting location at the beginning of the assay and their attraction to chemical odorants is evaluated as the nematodes distribute in the assay arena. Evaluation in this case consists of both scoring circles as well as each respective region: Region 1, Middle region, and Region 2.

  2. Jumping assay procedure

    Preparing jumping assay
    1. Drilling a 1.25 mm hole through the side of a 5 cm petri dish and lid to allow odors to be introduced with the lid on, thus preventing drying or the confounding effects of drafts (Figure 4).
    2. Place a 55 mm Whatman 1 filter paper on the bottom of the dish to absorb and retain moisture while providing a fibrous material to facilitate jumping. The filter paper acts as a soil-like substrate. Add < 100 nematodes suspended in ~200 μl of tap water.
    3. Introduce gases (e.g. 10% CO2) using a 10 ml Hamilton gastight syringe equipped with a Hamilton blunt needle (22 s/2”/3) (Figure 4).
    4. Introduce other odors (e.g. insect headspace or odorants) using a 10 ml Luer-Lok Becton Dickinson syringe with a Becton Dickinson needle (21 gauge 1.5”) with the end clipped to be blunt.

    Host-evoked jumping assay
    Nematodes in the genus Steinernema were first observed to be capable of jumping in Reed and Wallace (1965). This was the first observed instance of jumping among nematodes and certain species of Steinernema remain the only nematodes known to be capable of jumping. It was shown that S. carpocapsae can jump an average of 4.8 + 0.8 mm (nine times it’s body length) and an average height of 3.9 + 0.1 mm (seven times it’s body length) (Campbell and Kaya, 1999). Jumping is an unusual behavior for a soft-bodied, limbless, invertebrate.
    1. For host-evoked jumping, the number of hosts inside a syringe could vary, depending on the size of the host (Dillman et al., 2012).
    2. The attraction of the host headspace can also be tested without the presence of host-produced CO2 by adding a column of soda lime between the syringe and the needle. A six-inch column of Nalgene (8050-0250) FTP 3/16” OD tubing can be filled with 2-5 mm soda lime pellets (Figure 1C). The column is fitted on one end with fitting 731-8226, which accommodates the needle and on the other with fitting 731-8223 from a low-pressure fittings kit. The fittings can be wrapped in Teflon tape to ensure an airtight fit.

    Odor-evoked jumping assay
    1. For odorants, place a small piece of filter paper in the syringe and place 5 μl of undiluted odorant onto the filter paper. The control assay should be done using the same diluent that was used for the odorant (e.g. paraffin oil, water, or ethanol). Cover the hole drilled into the dish with parafilm and punctured only during assays.
    2. Only infective juveniles (IJs) of certain species of Steinernema insect-parasitic nematodes are known to jump (Campbell and Kaya, 2000). Add nematodes to the prepared jumping arena in liquid suspension such that ~100 nematodes are suspended in approximately 200 μl of water. Evenly distribute the nematode suspension on the filter paper. This assay can be used for single nematode jumping by placing a single nematode in the middle of the arena by pipetting through a pulled glass capillary. Pre-moisten the filter paper with 200 μl of tap water.
    3. To examine odor-evoked jumping, randomly select standing individual nematodes and present them, one at a time, with a small puff (~0.5 ml volume) of either air control or volatile stimulus. Only test individuals once during a standing bout. While it is possible for individual nematodes to jump more than once, often they will adhere to the lid of the plate after jumping and it is unlikely that the researcher will test a nematode that has already jumped.
      Introduce the odorant by slowly bringing the syringe needle tip to within ~2 mm of the standing individual and delivering a small puff of the mixture in the syringe (~0.5 ml volume) (Figure 4).
      Observe and record the behavioral changes of standing individuals within an 8 sec response interval after presenting the cue. Responses can be scored using a binary criterion of whether or nor the individual jumped or it can be divided into more categories if desired (Dillman et al., 2012).
    4. A normalized jumping index (J.I.) can then calculated. For stimuli that evoked higher levels of jumping than the control, the J.I. and SEM should be calculated as:


      For stimuli that evoked lower levels of jumping than the control, the J.I. and SEM should be calculated as:

      Calculating the response in this way will result in a normalized J.I. that ranges from -1 to +1.
    1. Approximately 20 IJs can be tested from the same arena. As with the chemotaxis assay, a soda lime column can be used to remove host-produced CO2. In the jumping assay a 2 inch column of Nalgene (8050-0250) FTP 3/16” OD tubing containing 2-5 mm soda lime pellets. This column can be attached to the syringe using fittings from a low-pressure fittings kit. Fitting 731-8226 can attach to the needle while the other end of the column is attached to the syringe with fitting 731-8223. That end should fit snugly onto the Luer-Lok end of the 50 ml syringe (Figure 1D). The fittings can be wrapped in Teflon tape to ensure an airtight fit. Alternatively the column can be placed between 2 female-ended Swagelok compression fittings of the appropriate size (Dillman et al., 2012).


      Figure 4. Jumping assay setup. A. A diagram of the jumping assay. A 5 cm petri dish and lid have a 1.25 mm hole drilled through the side to allow odors to be introduced with the lid on, thus preventing drying or the confounding effects of drafts. A 55 mm Whatman 1 filter paper is on the bottom of the dish to absorb and retain moisture while providing a fibrous material to facilitate jumping. B. The odorant is introduced by slowly bringing the syringe needle tip to within ~2 mm of the standing individual while a small puff of odorant is delivered (~0.5 ml volume).

Representative data

Video 1. Chemotaxis assay

Video 2. Jumping assay

Notes

These experiments can show some variability and are highly sensitive to changes in temperature, humidity, and the introduction of transient odors (e.g. perfume, cologne, soap, food, etc.) I have noticed that foods being cooked or eaten nearby, especially fragrant food such as popcorn, which smells like the attractant diacetyl, can affect chemotaxis and jumping behavior. Because of the high sensitivity and variability of these behavioral assays it is important that test runs and controls are run in parallel, using the same conditions. Having many replicates can help increase the behavioral signal.
Although behavioral experiments tend to have high sensitivity and variability, I have found that observed phenotypes using these techniques are robust over many days of experiments and between different batches of nematodes, both for jumping and chemotaxis, though there are occasional batches with unusual behavior.
The proclivity for chemotaxis assay is an interesting extension of the regular chemotaxis assay that provides more data and finer resolution into chemotaxis behavior. While previous work shows high chemotaxis indices for several species, which leads the reader to conclude that the species have similar chemoattractive preferences and abilities (e.g. Hallem et al. 2011; Dillman et al. 2012), a PCI assay might reveal differences in the proclivity for chemotaxis between these species. For example, whereas Steinernema glaseri and S. carpocapsae may both have a high CI toward waxworm odors, perhaps only 20% of the S. carpocapsae population being tested participates in the behavior while 80% of S. glaseri may be participating. The PCI is another way to explore the behavioral ecology of nematode olfactory responses.

Recipes

  1. Chemotaxis agar
    1.6% BBL-agar or 2% Difco-agar
    5 mM potassium phosphate (pH 6.0)
    1 mM CaCl2
    1 mM MgSO4

Acknowledgments

This laboratory protocol is a free adaptation of various published and unpublished protocols and has evolved over time (Ward, 1973; Bargmann et al., 1993; Campbell and Kaya, 2000; Hallem et al., 2011; Dillman et al., 2012; Castelletto et al., 2014). A.R.D. was funded by initial lab startup funds from the University of California, Riverside.

References

  1. Bargmann, C. I., Hartwieg, E. and Horvitz, H. R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74(3): 515-527.
  2. Campbell, J. F. and Kaya, H. K. (1999). How and why a parasitic nematode jumps. Nature 397: 485-486.
  3. Campbell, J. F. and Kaya, H. K. (2000). Influence of insect-associated cues on the jumping behavior of entomopathogenic nematodes (Steinernema spp.). Behavior 137: 591-609.
  4. Castelletto, M. L., Gang, S. S., Okubo, R. P., Tselikova, A. A., Nolan, T. J., Platzer, E. G., Lok, J. B. and Hallem, E. A. (2014). Diverse host-seeking behaviors of skin-penetrating nematodes. PLoS Pathog 10(8): e1004305.
  5. Dillman, A. R. and Sternberg, P. W. (2012). Entomopathogenic nematodes. Curr Biol 22(11): R430-431.
  6. Dillman, A. R., Guillermin, M. L., Lee, J. H., Kim, B., Sternberg, P. W. and Hallem, E. A. (2012). Olfaction shapes host-parasite interactions in parasitic nematodes. Proc Natl Acad Sci U S A 109(35): E2324-2333.
  7. Hallem, E. A., Dillman, A. R., Hong, A. V., Zhang, Y., Yano, J. M., DeMarco, S. F. and Sternberg, P. W. (2011). A sensory code for host seeking in parasitic nematodes. Curr Biol 21(5): 377-383.
  8. Reed, E. M. and Wallace, H. R. (1965). Leaping locomotion by an insect-parasitic nematode. Nature 206: 210-211.
  9. Ward, S. (1973). Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants. Proc Natl Acad Sci U S A 70(3): 817-821.

简介

线虫具有敏感的嗅觉感知,其用于检测和区分许多挥发性气味剂。 一些气味有吸引力,其他排斥,还有一些没有引起特别的反应。 趋化性测定可用于确定某些气味在许多不同行为中的作用,包括觅食,捕食者回避和配偶吸引。 除了趋化性之外,昆虫病原属Steinernema中的一些种类的线虫可以跳跃,这被认为在宿主寻找和分散中起重要作用(Dillman和Sternberg,2012)。 跳跃和趋化性测定已经成功地用于鉴定在多种线虫中刺激这些行为的气味剂(Bargmann等人,1993; Campbell和Kaya,1999; Hallem等人, 2011; Dillman等人,2012; Castelletto 等人,2014)。 这里基于文献的成长体提供了趋化性和跳跃测定的详细方案。

关键字:昆虫, 趋化性, 跳跃的, 行为, 线虫

材料和试剂

  1. 线虫
    注意:Caenorhabditis遗传学中心(CGC)是许多常用线虫的重要资源( http://cbs.umn.edu/cgc/home ),但并非所有线虫都可通过此资源使用。 例如,昆虫病原线虫(EPN)不能通过CGC获得,并且需要从现场取样收集或从另一个研究者的实验室请求。
  2. 要测试的所选气体百分比(例如 10%CO <2>)的天然气混合物,其余部分为N 2 2(Praxair, 目录号:7727-37-9)
  3. Parafilm(Thermo Fisher Scientific,目录号:13-374-10)
  4. 石蜡油
  5. Nalgene(Thermo Fisher Scientific,目录号:8000-0020)FTP 1/8"I.D。 管道
  6. 苏打石灰(2-5mm粒料)(Sigma-Aldrich,目录号:72073)
  7. ½英寸特氟隆胶带
  8. 激光打印机透明胶片(如 Apollo VCG7060E)
  9. 低压系统配件套件(Bio-Rad Laboratories,目录号:7318220)
  10. 叠氮化钠(Thermo Fisher Scientific,目录号:BP9221-500)
  11. BBL-琼脂(BD)或Difco-琼脂
  12. 100×15mm培养皿(VWR International,目录号:25384-088)
  13. 60×15mm培养皿(VWR International,目录号:25384-090)
  14. 1.55mm Whatman 1滤纸(Fisher Scientific,目录号:09-805B)
  15. Blunt Luer-Lok针(例如HAMPTON RESEARCH CORP 22s/2"/3)
  16. 一次性10ml 10ml Luer-Lok注射器(例如BD)
  17. 一次性针(21号,1.5英寸)(例如BD)
  18. 1/8英寸I.D. (内径)PVC管
  19. 50ml气密注射器(例如HAMPTON RESEARCH CORP,型号:1050)
  20. 10ml气密注射器(例如HAMPTON RESEARCH CORP,型号:1010)
  21. 趋化性琼脂(参见Recipes)

设备

  1. 解剖立体显微镜(例如,徕卡显微系统公司,型号:Leica M80)
  2. 输注注射泵(例如哈佛装置,型号:PHD 22/2000)
  3. 31号钻头(3.05毫米)(如 Alltrade Tools LLC,型号:480902旋转工具组)
  4. 56号钻头(1.18毫米)(如 Alltrade Tools LLC,型号:480902旋转工具组)
  5. Drill或dremmel(例如 Alltrade Tools LLC,型号:480902旋转工具套件)
  6. 防震平台(泡沫底有纸板平台)

程序

  1. 趋化性测定程序

    准备趋化性测定
    1. 在10厘米的培养皿盖上钻两个3.05毫米的孔,每个孔应该 沿着直径从边缘1cm,正好彼此相对。 为每个测定准备一个盖子,将同时进行,但是 一旦准备好,盖子可以重复使用。
    2. 连接柔性PVC管 (1/8英寸内径)到每个孔的顶部,用管 稍微突出到盖子的内部(图1A)。 结尾 进入板盖的PVC管可以用多层包裹 parafilm以确保紧密贴合。 管道的另一端将是 连接到50ml气密注射器(图1B)。底部的 测定板应该具有附加的评分模板(图2)。
    3. 刻痕模板可以印刷在透明胶片上并附着  到使用小条双面胶带的测定板。评分 区域是沿着板的每一侧上的2cm直径的圆 直径,圆的中心距离板的边缘1cm。
    4. 线虫可以通过首先加入1-2μl的20%TritonX-100来制备 的水溶液加入到其中含有它们的瓶中。的 线虫可以通过放置5个10μl的滴定量 培养皿盖。可以计数存在的线虫的密度 可以计算溶液中的线虫。足够的体积 获得所需的线虫数量,然后可以放入 Eppendorf管,并允许休息2分钟或更长时间。线虫会  沉到底部,并且可以吸出过量的水。颗粒  然后可以通过移液管提取蠕虫。

    气体趋化性测定
    1. 在开始测定之前,立即填充50ml气密 注射器根据实验。 例如,如果评估 吸引10%CO 2,一个注射器将填充10%CO 2,同时   其他注射器将充满空气控制。 注射器将   使用注射泵(例如PHD)以0.5ml/min的速率降压 22/2000,Harvard Apparatus)。
    2. 地点   大约5-10微升含有约50-150个线虫的蠕虫小球 测定板的中心。 这些线虫应该是阶段匹配的 或者尽可能同步。 不同的生命阶段已知改变   它们的气味偏好(Hallem等人,2011)。 每个板应该 不受干扰 - 在抗振平台上 - 持续时间 测定。 这些测定可以根据不同的长度运行 进行特定实验。 一小时似乎是标准,但是 已经显示10,20,40和60分钟可以产生相同的结果 在一些实验中(Hallem等人,2011)。

    主机趋化性测定
    1. 可以通过将多个宿主放置在50ml内来测试宿主顶空   气密注射器; 该数量将根据主机大小而变化。
    2. 主机顶部空间的吸引力也可以无需测试 通过添加一列钠石灰到主体产生的CO 2的存在 测定装置(图1C-D)。 一个6英寸的Nalgene(8050-0250)柱 3/16"ID管可以填充2-5 mm钠石灰颗粒(图1C)。 该柱的一端用可附接的配件731-8226安装  到在另一端上运行到测定板盖的管道 从低压连接到配件731-8223 配件套件。那一端应该紧贴在50的Luer-Lok末端  ml注射器(图1D)。配件可以用特氟隆胶带包裹 确保气密配合。
    3. 在测定结束时,数目 应计数板的每一侧上的记分区域中的蠕虫  趋化性指数应计算为:

      在该实施例中,测试的气味剂是CO 2,而室内空气是对照,但是相同的计算将用于其它气味剂,尽管控制可能不同。只有当≥3个蠕虫进入评分区域时,才应对分析进行评分,并且应当舍弃所有蠕虫表现出相同方向偏差的测定期的试验。
    1. 所述测定意欲用作群体测定 〜50-150线虫在实验中,但可以用于单线虫 实验。

    气味趋化性测定
    1. 对于气味趋化性测定,将1μl的1M叠氮化钠放入 中心作为麻醉剂。 放置5微升的气味   一个评分区域的中心,同时放置5μl的对照 (石蜡油,dH 2 O或乙醇)在另一个的中心 评分区。
    2. 与气体测定,放置〜2微升的蠕虫 在测定板的中心含有〜50-150个线虫的沉淀。 离开测定板不受干扰,并在1小时后(对于 宿主和CO 2趋化性测定)或3小时(用于气味测定)。 一个 可以在不使用叠氮化钠的情况下进行替代测定 在分配的实验时间之后立即对测定板进行评分  已经过去了。气味剂测定也可以在没有麻醉剂的情况下进行, 但是然后在实验后立即需要对板进行评分  完成。


      图1.趋化性测定设置。 A.图片10 cm测定板盖,其中具有两个3.05mm孔。每个孔距离1厘米 沿着直径的边缘,恰好彼此相对。 1/8英寸 ID。 PVC管包裹在石蜡膜中,然后装入孔中。 B.A  1/8英寸管道的图片连接到气密注射器。 C.A 苏打石灰柱的图片,使用Nalgene(8050-0250) FTP 3/16"I.D.管道在两端有低压配件。在里面 图片,该列为空,但在测定中将填充2-5 mm钠石灰颗粒。 D.附着的钠钙柱的图片 到气密注射器和连接到测定的PVC管 板盖

      图2.用于对趋化性测定进行评分的模板。线虫放置在起始位置 测定和它们对化学气味剂的吸引力被评价为 线虫在测定领域中分布。

    趋化性测定的倾向性
    1. 作为趋化性测定的延伸,可以是替代模板 用于对趋化性指数和a的线虫评分 趋化性指数(PCI)的倾向性。 PCI模板(图3)可以 在透明纸上打印并连接到测定板上 双面胶带。
    2. 在评分期间发现的线虫数量   在每个圆应该记录为正常。 然后,每个区域应该 并记录每个区域中的线虫数量。
    3. 然后可以使用这些数据计算每个区域的总体百分比。


      图3.用于对两者的趋化性测定进行评分的模板 趋化性指数(CI)和趋化性指数的倾向性。线虫是   放置在测定开始的起始位置和它们   对化学气味剂的吸引力被评价为线虫 分布在测定领域。 在这种情况下的评价由两者组成 得分圈以及每个相应的区域:区域1,中间 区域和区域2.

  2. 跳跃测定程序

    准备跳跃测试
    1. 在5cm培养皿和盖子的侧面钻一个1.25mm的孔   允许在盖子上引入气味,从而防止干燥或   草案的混杂效应(图4)。
    2. 放置55 mm Whatman 1滤纸在底部的吸收和保留 水分,同时提供纤维材料以促进跳跃。 的 滤纸充当土壤状底物。 添加< 100线虫 悬浮在〜200μl自来水中
    3. 引入气体(例如 10%CO 2 )   使用装备有Hamilton钝器的10ml Hamilton气密注射器 针(22s/2"/3)(图4)。
    4. 使用10ml Luer-Lok Becton Dickinson引入其他气味(例如昆虫顶空或气味剂) 注射器用Becton Dickinson针(21规格1.5")与末端 削减钝。

    主机诱发的跳跃测试
    首次观察到属于Steinernema的线虫能够在Reed和Wallace(1965)中跳跃。这是在线虫中首次观察到的跳跃的实例,并且某些种类的斯坦内马马仍然是已知能够跳跃的唯一线虫。它显示, (平均身高的9倍),平均高度为3.9 + 0.1mm(身体长度的7倍)(Campbell和Kaya,1999)。跳跃是一个不寻常的行为,一个柔软,无肢,无脊椎动物
    1. 对于主机诱发的跳跃,注射器内的主机数量 根据宿主的大小而变化(Dillman等人,2012)。
    2. 主机顶部空间的吸引力也可以无需测试 存在主体产生的CO 2,通过在其间加入钠钙柱 注射器和针。 一个6英寸的Nalgene(8050-0250)柱   3/16"外径管可以填充2-5毫米的钠钙颗粒(图 1C)。 该柱的一端用配件731-8226装配 容纳针和在另一个与配件731-8223从a 低压配件工具包。 配件可以用特氟隆胶带包裹   确保气密配合。

    气味诱发跳跃测定
    1. 对于气味剂,将一小块滤纸放在注射器中 将5μl未稀释的气味剂放在滤纸上。 对照测定   应使用与用于气味剂的稀释剂相同的稀释剂进行 (例如石蜡油,水或乙醇)。 盖上钻进的孔 皿与石蜡膜和穿孔只在测定期间
    2. 只要 已知某些种类的Steinernema昆虫寄生线虫的感染性幼体(IJ)会跳跃(Campbell和Kaya,2000)。 添加线虫到准备的跳跃竞技场在液体悬浮液中 将约100条线虫悬浮于约200μl水中。 均匀地将线虫悬浮液分布在滤纸上。这个 测定可以通过放置单个线虫用于单线虫跳跃 在竞技场中间的线虫通过吸取通过被拉扯的玻璃 毛细管。用200μl自来水预先过滤滤纸。
    3. 为了检查气味诱发跳跃,随机选择站立的个人 线虫,并提出他们,一次一个,用小吹(〜0.5毫升 体积)的空气控制或挥发性刺激。只有测试 个人一次在站立开始。虽然它是可能的 单个线虫跳不止一次,往往他们会坚持 板的盖子跳后,这是不可能的 研究者将测试已经跳过的线虫 介绍 通过慢慢地将注射器针尖在〜2mm内的气味  站立的个体和递送混合物的小泡沫 注射器(〜0.5ml体积)(图4)。
      观察并记录 站立个体在8秒响应内的行为变化 间隔后呈现提示。响应可以使用a 二元判断是否是个人跳跃还是可以 如果需要,分成更多类别(Dillman等人,2012)
    4. 一个  然后可以计算归一化跳跃指数(J.I.)。对于刺激 诱发的跳跃水平高于对照组。和SEM 应计算为:


      对于诱发比对照更低水平的跳跃的刺激,J.I。和SEM应计算为:

      以这种方式计算响应将导致归一化的J.I.范围从-1到+1。
    1. 大约20 IJ可以从同一个舞台测试。和 趋化性测定,钠钙柱可用于去除宿主产生的  CO 2。在跳跃测定中,2英寸Nalgene柱(8050-0250)FTP 3/16"OD管,包含2-5mm钠石灰颗粒。此列可以 使用从低压配件套件的配件连接到注射器。  接头731-8226可以连接到针头,而另一端 柱用配件731-8223连接到注射器。那应该结束  紧密贴合在50ml注射器的Luer-Lok末端(图1D)。的 配件可以包裹在特氟龙胶带中,以确保气密配合。 或者,柱可以放置在2个女性端的Swagelok之间 适当尺寸的压缩配件(Dillman等人,2012)。


      图4.跳跃测定设置。A.跳跃测定图。 A 5 cm培养皿和盖子有一个1.25毫米的孔穿过侧面到 允许在盖子上引入气味,从而防止干燥或 草案的混杂效应。打开55 mm Whatman 1滤纸 在底部吸收和保留水分,同时提供一个 纤维材料以方便跳跃。引入气味剂 慢慢地将注射器针尖放置在约2毫米的站立 个体,同时递送少量的气味物质(〜0.5ml体积)。

代表数据

视频1.趋化性测定
<! - [if!IE] <! - <![endif] - >

要播放视频,您需要安装较新版本的Adobe Flash Player。

获取Adobe Flash Player

<! - [if!IE]> - >
<! - <![endif] - >

视频2.跳跃测试
<! - [if!IE]> - <! - <![endif] - >

要播放视频,您需要安装较新版本的Adobe Flash Player。

获取Adobe Flash Player

<! - [if!IE]> - >
<! - <![endif] - >

笔记

这些实验可以显示一些变化性,并且对温度,湿度的变化以及瞬时气味的引入(例如香水,古龙水,肥皂,食物,等等)是非常敏感的。 我注意到,在附近烹饪或食用的食物,特别是香味食物,如爆米花,闻起来像引诱剂丁二酮,可以影响趋化性和跳跃行为。由于这些行为测定的高灵敏度和可变性,重要的是测试运行和对照使用相同的条件并行运行。有许多重复可以帮助增加行为信号。
虽然行为实验倾向于具有高灵敏度和变异性,但我发现使用这些技术的观察到的表型在许多天的实验和在不同批次的线虫之间,对于跳跃和趋化性是稳健的,尽管偶尔具有异常行为的批次。 br /> 趋化性测定的倾向是常规趋化性测定的有趣延伸,其提供更多数据和更好的分辨率到趋化性行为。虽然先前的工作显示了几种物种的高趋化性指数,这导致读者得出结论,物种具有相似的化学吸引偏好和能力(例如2011年; Hallem等人2011; Dillman& 2012),PCI测定可能揭示这些物种之间趋化性的倾向性的差异。例如, glaseri carpocapsae 可能都具有高CI对蜡虫气味,也许只有20%的。 carpocapsae 群体参与行为,而80%的 S。 glaseri 可能正在参与。 PCI是探索线虫嗅觉反应的行为生态学的另一种方式。

食谱

  1. 趋化性琼脂
    1.6%BBL-琼脂或2%Difco-琼脂 5mM磷酸钾(pH6.0) 1mM CaCl 2
    1mM MgSO 4

致谢

该实验室方案是对各种已公开和未公开的方案的自由适应,并且随着时间演变(Ward,1973; Bargmann等,1993; Campbell和Kaya,2000; Hallem等, ,2011; Dillman等人,2012; Castelletto 等人,2014)。 A.R.D. 由来自加州大学河滨分校的初始实验室启动资金资助。

参考文献

  1. Bargmann,C.I.,Hartwieg,E。和Horvitz,H.R。(1993)。 气味选择性基因和神经元在C中介导嗅觉。 elegans 。 74(3):515-527
  2. Campbell,J.F。和Kaya,H.K。(1999)。 寄生线虫的跳跃方式和原因。 自然 397:485-486
  3. Campbell,J.F。和Kaya,H.K。(2000)。 昆虫相关线索对昆虫病原线虫跳跃行为的影响(Steinernema&/em> spp。) 行为 137:591-609
  4. Castelletto,M.L.,Gang,S.S.,Okubo,R.P.,Tselikova,A.A.,Nolan,T.J.,Platzer,E.G.,Lok,J.B.and Hallem,E.A。(2014)。 皮肤穿透性线虫的不同宿主寻求行为。 /em> 10(8):e1004305。
  5. Dillman,A.R。和Sternberg,P.W。(2012)。 昆虫致病性线虫。Curr Biol 22(11): R430-431。
  6. Dillman,A.R.,Guillermin,M.L.,Lee,J.H.,Kim,B.,Sternberg,P.W.and Hallem,E.A。(2012)。 嗅觉在寄生线虫中形成寄主寄生虫相互作用。 Proc Natl Acad Sci USA 109(35):E2324-2333。
  7. Hallem,E.A.,Dillman,A.R.,Hong,A.V.,Zhang,Y.,Yano,J.M.,DeMarco,S.F.and Sternberg,P.W。(2011)。 主机寻求的感觉代码 寄生线虫。 Curr Biol 21(5):377-383。
  8. Reed,E.M。和Wallace,H.R。(1965)。 通过昆虫寄生线虫跳跃运动。 Nature 206:210-211
  9. Ward,S。(1973)。 线虫Caenorhabditis elegans的趋化性: :引诱物的鉴定和分析 通过使用突变体的反应。 Proc Natl Acad Sci USA 70(3):817-821。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Baiocchi, T. and Dillman, A. R. (2015). Chemotaxis and Jumping Assays in Nematodes. Bio-protocol 5(18): e1587. DOI: 10.21769/BioProtoc.1587.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。