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Aphids are phloem-feeding insects that successfully colonize specific host plant species. Aphid performance on a given plant is commonly measured by assessing fecundity of an aphid species that is adapted to the host. However, this approach may not reveal roles for plant genes in defense pathways that adapted aphids suppress. The host range of the pea aphid (Acyrthosiphon pisum) is mostly restricted to plants of the legume family, and does not include Arabidopsis (Arabidopsis thaliana). Pea aphids die within a few days of being placed on Arabidopsis plants, and their survival therefore provides a sensitive measure of the status of the host plant defenses. This protocol describes how to measure the survival rate of the pea aphid on the non-host plant Arabidopsis. The protocol consists of two phases: first, obtaining a population of pea aphids of synchronized age; and secondly measuring their survival on Arabidopsis plants.

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Pea Aphid Survival Assays on Arabidopsis thaliana
拟南芥的豌豆蚜存活实验

植物科学 > 植物免疫 > 病害生物测定
作者: David C. Prince
David C. PrinceAffiliation: Department of Cell and Development Biology, John Innes Center, Norwich Research Park, Norwich, UK
Bio-protocol author page: a1683
Sam T. Mugford
Sam T. MugfordAffiliation: Department of Cell and Development Biology, John Innes Center, Norwich Research Park, Norwich, UK
Bio-protocol author page: a1684
Thomas R. Vincent
Thomas R. VincentAffiliation: Department of Metabolic Biology, John Innes Center, Norwich Research Park, Norwich, UK
Bio-protocol author page: a1685
 and Saskia A. Hogenhout
Saskia A. HogenhoutAffiliation: Department of Cell and Development Biology, John Innes Center, Norwich Research Park, Norwich, UK
For correspondence: saskia.hogenhout@jic.ac.uk
Bio-protocol author page: a1686
Vol 4, Iss 19, 10/5/2014, 2880 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1251

[Abstract] Aphids are phloem-feeding insects that successfully colonize specific host plant species. Aphid performance on a given plant is commonly measured by assessing fecundity of an aphid species that is adapted to the host. However, this approach may not reveal roles for plant genes in defense pathways that adapted aphids suppress. The host range of the pea aphid (Acyrthosiphon pisum) is mostly restricted to plants of the legume family, and does not include Arabidopsis (Arabidopsis thaliana). Pea aphids die within a few days of being placed on Arabidopsis plants, and their survival therefore provides a sensitive measure of the status of the host plant defenses. This protocol describes how to measure the survival rate of the pea aphid on the non-host plant Arabidopsis. The protocol consists of two phases: first, obtaining a population of pea aphids of synchronized age; and secondly measuring their survival on Arabidopsis plants.

[Abstract]

Materials and Reagents

  1. 3 to 4-week-old Vicia faba plants grown in 14 h light (18 °C)/10 h dark (15 °C)
  2. 7-week-old Arabidopsis plants grown in a short day condition (10 h light/14 h dark (22 °C)
  3. Pea Aphids (Acyrthosiphon pisum) maintained on Vicia faba plants grown in 14 h light (18 °C)/10 h dark (15 °C)

Equipment

  1. Plant growth facilities
  2. Insect-proof cage to contain Vicia faba plants (Note 1)
  3. Petri dish
  4. Moist artist’s paintbrush (size 2 or 4)
  5. Clip cages (Figure 1)
  6. Plant labels
  7. Marker pen


    Figure 1. Clip cage. Composed of a metal double prong hair clip (50 mm long), two pieces of plastic tube (10 and 5 mm high, 2 mm thick, 25 mm diameter), two circles of felt (25 mm diameter, 4 mm across, 1 mm thick), and two pieces of fine gauze (25 mm diameter). The hair clip is heated and pushed into the plastic tubes. The felt and gauze are attached with superglue. Scale bar = 5 mm

Software

  1. Microsoft® Excel

Procedure

  1. Obtaining a cohort of aged pea aphids
    1. Collect adult pea aphids in a petri dish from a stock cage (Note 2).
    2. Use a moist paintbrush to transfer the aphids to a fresh cage containing several Vicia faba plants in pots.
    3. Place the cage, containing the aphids and plants, in long day growth conditions [14 h light (22 °C), 10 h night (15 °C)].
    4. After 24 h remove all the adult aphids, while the nymphs remain (Note 3).
    5. Allow the nymphs to reach the adult stage (Note 4).

  2. Setting up the experiment
    1. Use plastic plant labels to label 7-week-old Arabidopsis with their genotype and the plant number (e.g. Col-0, plant 1). Use at least five plants per genotype per replicate.
    2. Collect the cohort of aged pea aphids in a petri dish.
    3. Use a moist paintbrush to transfer 5 of the aphids to a clip cage.
    4. Gently clip the cage onto a fully developed leaf of the Arabidopsis plant.
    5. Repeat the previous two steps until all the plants have one clip cage each.
    6. Randomize the plants across the area that they are to be grown in (Note 5).
    7. Place the plants in short day conditions [8 h light (18 °C), 16 h night (16 °C)].

  3. Collecting the results
    1. The day that the aphids are caged to the Arabidopsis plants is day 0.
    2. Count and record the number of live adults in the clip cages on days 2 to 7.

      Table 1. Example data from one repeat of an experiment with Col-0 Arabidopsis, with readings taken on days 3 to 7
      Number of adult aphids alive
      Day   
      Plant 1  
      Plant 2   
      Plant 3   
      Plant 4   
      Plant 5
      3
      4
      5
      4
      5
      5
      4
      4
      2
      3
      1
      3
      5
      0
      2
      1
      0
      0
      6
      0
      0
      0
      0
      0
      7
      0
      0
      0
      0
      0

  4. Repetition
    1. Repeat the whole experiment (steps 1-3) on at least 3 separate occasions.

  5. Analysing the results
    1. Calculate the time point at which survival on wild type Arabidopsis is 50% (Note 6). If this occurs between two of the days (e.g. between day 3 and 4) then average the number of adults alive on these two days.
    2. Use this time point to calculate the number of adults alive on each plant (Note 7).
    3. Analyze by classical linear regression analysis using a Poisson distribution within a generalized linear model.

Notes

  1. The cage should be large enough for the Vicia faba plants to grow, and allow sufficient light and ventilation while preventing the aphids from escaping. We use Perspex cages (47 cm high x 54 cm deep x 24 cm wide) with a fine mesh covering attached by magnetic strips at one end to serve as a door.
  2. 5 to 10 pea aphids per Arabidopsis plant in the experiment should be sufficient, as approximately one nymph is born per adult in 24 h at 22 °C. The aphids reproduce slower at lower temperatures and therefore more adults are needed to produce the same number of nymphs.
  3. Make sure to count the number of adults added to the cage, and remove the same number of adults at this stage.
  4. In the conditions stated, pea aphids should reach the adult stage by 8 or 9 days, having undergone four moults. Only adult aphids reproduce. It is recommended to use aphids 10 to 14 days old.
  5. For example, if you are placing the experimental plants in 3 trays for the experiment then randomly assign each plant a number of 1, 2 or 3, corresponding to trays 1, 2 and 3. This can be done using the RANDBETWEEN function in Microsoft® Excel.
  6. Average the number of adult pea aphids alive on each day across all your repeats of the wild type Arabidopsis. Then plot this data against the experiment day number on a line graph. See Reference 1 (Prince et al., 2014) Figure 6B for an example.
  7. For example, the data in Table 1 is part of a larger data set where 50% survival is between day 3 and 4. Therefore, the data would be calculated as: Plant 1 = 4, Plant 2 = 3.5, Plant 3 = 3.5, Plant 4 = 3, Plant 5 = 4.

Acknowledgements

We thank Andrew Davis for the photograph of the clip cage. This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. BB/J004553/1 to S. A. H.), the John Innes Foundation (to S. A. H.), a Biotechnology and Biological Sciences Research Council studentship (to D. C. P.) and a John Innes Foundation studentship (to T. R. V). This protocol is adapted from Reference 1 (Prince et al., 2014).

References

  1. Prince, D. C., Drurey, C., Zipfel, C. and Hogenhout, S. A. (2014). The leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the cytochrome P450 PHYTOALEXIN DEFICIENT3 contribute to innate immunity to aphids in Arabidopsis. Plant Physiol 164(4): 2207-2219.

材料和试剂

  1. 在14小时光照(18℃)/10小时黑暗(15℃)下生长的3至4周龄的蚕豆植物植物
  2. 在短日照条件(10小时光照/14小时黑暗(22℃))中生长的7周龄拟南芥植物
  3. 在14小时光照(18℃)/10小时黑暗(15℃)下生长的蚕豆蚜(绿豌豆豌豆)维持在蚕豆

设备

  1. 植物生长设施
  2. 含有蚕豆植物(注1)的防虫笼子
  3. 培养皿
  4. 潮湿艺术家的画笔(尺寸2或4)
  5. 夹笼(图1)
  6. 植物标签
  7. 记号笔


    图1.夹笼。由金属双头发夹(50毫米长),两片塑料管(10和5毫米高,2毫米厚,25毫米直径),两个圆圈 的毡(25mm直径,4mm跨,1mm厚)和两片细纱(直径25mm)。 将发夹加热并推入塑料管中。 毛毡和纱布附着超强力胶。 比例尺= 5 mm

软件

  1. Microsoft ® Excel

程序

  1. 获得一组老豌豆蚜虫
    1. 从储存笼中的培养皿中收集成虫豌豆蚜虫(注2)。
    2. 使用湿润的油漆刷将蚜虫转移到一个新鲜的笼子里,包含几个盆栽的蚕豆植物。
    3. 将含有蚜虫和植物的笼子放在长日照生长条件下[14小时光照(22℃),10小时夜晚(15℃)]。
    4. 24小时后,除去所有的成年蚜虫,而仙女留下(注3)。
    5. 允许若虫到达成年阶段(注4)。

  2. 设置实验
    1. 使用塑料植物标签标记7周龄拟南芥 基因型和植物数目(例如植物1的Col-0)。 至少使用五个 每个基因型每个重复的植物
    2. 在培养皿中收集老年豌豆蚜虫的群体。
    3. 使用湿润的油漆刷将5只蚜虫转移到夹子笼中。
    4. 将笼子轻轻地夹在拟南芥植物的完全发育的叶子上。
    5. 重复前两个步骤,直到所有植物都有一个夹笼。
    6. 将植物随机分布在他们生长的地区(注释5)。
    7. 将植物放置在短日照条件下[8小时光照(18℃),16小时夜(16℃)]。

  3. 收集结果
    1. 蚜虫被捕获到拟南芥植物的那一天是0. 0.
    2. 在第2至7天计算并记录夹子笼中的活成人数量。

      表1.来自使用Col-0 拟南芥 的实验的一次重复的实施例数据,/strong>
      成年蚜虫数量
      日   
      工厂1 
      工厂2   
      Plant 3    
      工厂4   
      植物5
      3
      4
      5
      4
      5
      5
      4
      4
      2
      3
      1
      3
      5
      0
      2
      1
      0
      0
      6
      0
      0
      0
      0
      0
      7
      0
      0
      0
      0
      0

  4. 重复
    1. 重复整个实验(步骤1-3)至少3次不同的场合。

  5. 分析结果
    1. 计算野生型拟南芥上存活的时间点 50%(注6)。 如果这发生在两天之间(例如在第3天之间)   和4)然后平均在这两天活着的成年人的数量。
    2. 使用此时间点计算每株植物活着的成虫数量(注7)。
    3. 通过在广义线性模型内使用泊松分布的经典线性回归分析。

笔记

  1. 笼子应该足够大以使蚕豆植物生长,并且允许足够的光和通风,同时防止蚜虫逃逸。 我们使用Perspex笼(47厘米高×54厘米深×24厘米宽),具有细小的网状覆盖物,通过磁条在一端作为门。
  2. 实验中5至10个豌豆蚜虫每个拟南芥植物应该是足够的,因为在22℃下在24小时内每个成年体出生约1个若虫。 蚜虫在较低温度下繁殖较慢,因此 需要更多的成年人来产生相同数量的若虫
  3. 确保计算添加到笼子里的成年人数,并在此阶段删除相同数量的成年人。
  4. 在所述条件下,豌豆蚜虫应达到成熟阶段8或9天,经历了4次蜕皮。只有成虫蚜虫繁殖。建议使用蚜虫10至14天。
  5. 例如,如果您将实验植物放置在3个托盘中进行实验,则随机地为每个植物分配1,2或3个对应于托盘1,2和3的数。这可以使用Microsoft中的RANDBETWEEN函数sup>® Excel。
  6. 平均每天在所有重复的野生型拟南芥中存活的成年豌豆蚜虫的数量。然后在线图上将此数据与实验日数绘图。参见参考文献1(Prince ,2014)图6B的示例。
  7. 例如,表1中的数据是较大数据集的一部分,其中50%的存活在第3天和第4天之间。因此,数据将计算为:植物1 = 4,植物2 = 3.5,植物3 =工厂4 = 3,工厂5 = 4

致谢

我们感谢安德鲁·戴维斯为夹子的照片。 这项工作是由生物技术和生物科学研究委员会(授予号BB/J004553/1至SAH),约翰·因尼斯基金会(SAH),生物技术和生物科学研究委员会学生计划(对DCP)和约翰·内斯 基础学生(至TR V)。 该协议改编自参考文献1(Prince ,,2014)。

参考文献

  1. Prince,D.C.,Drurey,C.,Zipfel,C.and Hogenhout,S.A。(2014)。 富含亮氨酸的重复受体样激酶BRASSINOSTEROID INSENSITIVE1相关激酶1和细胞色素P450 PHYTOALEXIN DEFINIENT3贡献 在拟南芥中对蚜虫的先天免疫。植物生理学 164(4):2207-2219。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Prince, D. C., Mugford, S. T., Vincent, T. R. and Hogenhout, S. A. (2014). Pea Aphid Survival Assays on Arabidopsis thaliana. Bio-protocol 4(19): e1251. DOI: 10.21769/BioProtoc.1251; Full Text
  2. Prince, D. C., Drurey, C., Zipfel, C. andHogenhout, S. A. (2014). The leucine-rich repeat receptor-like kinaseBRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the cytochrome P450PHYTOALEXIN DEFICIENT3 contribute to innate immunity to aphids in Arabidopsis. Plant Physiol 164(4): 2207-2219.




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