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Sep 2019
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Insect Feeding Assays with Spodoptera exigua on Arabidopsis thaliana
甜菜夜蛾对拟南芥的取食试验   

Yanrong YouYanrong You*Chunpeng AnChunpeng An*Chuanyou   LiChuanyou Li  (*共同第一作者)
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Abstract

Plant-insect interaction is an important field for studying plant immunity. The beet armyworm, Spodoptera exigua, is one of the best-known agricultural pest insects and is usually used to study plant interactions with chewing insects. Here, we describe a protocol for insect feeding assays with Spodoptera exigua lavae using model host plant Arabidopsis thaliana, which is simple and easy to conduct, and can be used to evaluate the effect of host genes on insect growth and thus to study plant resistance to chewing insects.

Keywords: Plant-insect interaction (植物-昆虫相互作用), chewing insects (咀嚼式昆虫), Spodoptera exigua (甜菜夜蛾), Arabidopsis thaliana (拟南芥), Insect feeding assay (昆虫喂食实验)

Background

Plants face a variety of biotic stresses throughout their lives, such as herbivore attack and pathogen infection. The beet armyworm, Spodoptera exigua, is a worldwide phytophagous pest with a broad host range, damaging various vegetable crops and causing considerable economic agricultural losses (Howe and Jander, 2008; Hu et al., 2013). Spodoptera exigua larvae usually feed on both foliage and fruit, and are extremely destructive. Arabidopsis thaliana is a host plant of Spodoptera exigua, and is also a classic model plant to study plant resistance to herbivores. Here, we describe a method adapted from our previous work (You et al., 2019) to conduct the feeding assays with Spodoptera exigua larvae on Arabidopsis thaliana rosette leaves. By counting the weight of the larvae after feeding leaves from different genotypes, we were able to evaluate plant resistance to herbivore attacks in the laboratory settings.

Materials and Reagents

  1. Square Petri dishes (100 mm × 100 mm, Beijing Ruiaizhengte Biological Technology Co., Ltd., catalog number: YC-HC99050 )
  2. Petri dishes (150 mm, Corning, catalog number: 430599 )
  3. 1.5 ml microtubes (AXYGEN, catalog number: MCT-150-C )
  4. Pipet tips (AXYGEN, catalog number: T-200-Y and T-1000-B )
  5. Micropore tape (3M, MicroporeTM, catalog number: 1530C-0 )
  6. Parafilm ( Bemis, catalog number: PM-996 )
  7. Toothpick (Suncha, catalog number: YQ1250 )
  8. Black cloth (Beiyang, catalog number: 13000133 )
  9. Arabidopsis thaliana
  10. Spodoptera exigua (KEYUN) (https://item.taobao.com/item.htm?spm=a1z09.2.0.0.10672e8dnlXuwi&id=567208183626&_u=pmk8luh1123)
  11. Artificial diet (Ingredients: wheat germ, yeast, carrageenan, konjac powder, sorbic acid, vitamin C, corn oil, and linoleic acid; KEYUN)
    (https://item.taobao.com/item.htm?spm=a1z09.2.0.0.10672e8dnlXuwi&id=43498077050&_u=pmk8luh74e5)
  12. Murashige & Skoog basal medium with vitamins (Phyto Technology Laboratories, catalog number: M519 )
  13. Bacto-agar (BD, BactoTM, catalog number: 214010 )
  14. 10% Bleach (KAO, 600 ml)
  15. 1 M KOH solution (Aladdin, catalog number: P112281 )
  16. Sucrose (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10021418 )
  17. Diethyl ether (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10009328 )
  18. Sterile distilled water
  19. Nutritional soil (moss peat:vermiculite = 2:1, PINDSTRUP, type: 0-10 mm)
  20. ½ MS medium (see Recipes)
  21. 0.8% agar medium (see Recipes)

Equipment

  1. Pipettes (Gilson, Pipetman® G)
  2. Graduated cylinder
  3. Reagent bottle
  4. Refrigerator or a cold room
  5. Tweezers
  6. Plant growth chamber
  7. Square pot
  8. Autoclave
  9. Laminar flow hood
  10. Balance
  11. Canon camera
  12. Ruler

Software

  1. Microsoft Excel

Procedure

  1. Cultivation of Arabidopsis thaliana plants
    1. Prepare the ½ MS solid medium, and pour enough media into square Petri dishes (100 mm × 100 mm) to cover approximately half of the depth of the dish (40 ml).
    2. Surface-sterilize Arabidopsis seeds in 1.5 ml microtubes by soaking in 10% bleach for 15 min, then remove all bleach residue by rinsing five times with sterile distilled water.
    3. Sow the seeds in the square Petri dishes containing ½ MS medium (Figure 1A), and seal the dishes with Micropore tape.
    4. Place the dishes in the dark at 4 °C for 2 days to allow for efficient and synchronous germination.
    5. Transfer the dishes to the growth chamber set at 22 °C with a 10-h light/14-h dark photoperiod for 10 days.
    6. Transplant the seedlings into square pots filled with nutritional soil (Figure 1B), and keep them in the growth chamber (22 °C, 10-h light/14-h dark) for another 4 weeks (Figure 1C).
      Note: Be careful not to damage the roots of the seedlings. Take care of the plants to prevent them from being affected by herbivores or pathogens.


      Figure 1. The cultivation of Arabidopsis plants. A. Sterilized Arabidopsis seeds were sowed on the square dish containing ½ MS medium. B. Ten-day old Arabidopsis seedlings were transplanted into a pot filled with nutritional soil. C. Arabidopsis seedlings grown in pot for 4 weeks are used for insect feeding assays.

  2. Preparation of Spodoptera exigua larvae
    1. Place the Spodoptera exigua eggs in a square Petri dish, and seal the dish with Micropore tape (Figure 2A).
    2. Place the dish in the 27 °C incubator with relative humidity of 40-50% for hatching (about 2-3 days).
    3. As soon as the eggs hatch, add artificial diet to the dish (Figure 2B) and continue incubation for 5 days.
      Note: Cut the artificial diet into small pieces of 1-1.5 cm square and placed them in the Petri dish at a certain interval from the eggs or larvae. Usually, after 5 days of feeding, the larvae become third-instar larvae.


      Figure 2. The hatching of Spodoptera exigua eggs. A. Spodoptera exigua eggs. B. Newly hatched larvae and the artificial diet.

    4. Use a toothpick to transfer the larvae into a new Petri dish (Video 1 and Figure 3A) and starve them for 12 h before being used in the experiments (Figure 3B).
      Note: This step needs to be careful and gentle. Usually, we use a water-soaked toothpick to let the larvae catch and gently transfer the larvae to prevent them from being injured.


      Figure 3. The starvation treatment of Spodoptera exigua larvae. A. A larva clutching a toothpick. B. Third-instar larvae starved for 12 h.

      Video 1. How to catch a larva with a toothpick?

  3. Insect feeding assays
    1. Prepare and pour enough 0.8% agar into plastic Petri dishes (150 mm) to cover approximately half of the depth of the dish.
    2. Cut mature rosette leaves of similar size from Arabidopsis plants grown in soil for 4 weeks, and place them in a plastic Petri dish (150 mm) containing 0.8% agar (Figure 4A).
      Note: If the leaves of different genotypes are similar in shape and size, we usually arrange them in the way shown in Figure 4A. And if the shape and size of the leaves vary greatly, it is required to weigh the leaves each time to ensure that the same weight of leaves are added.
    3. Use a water-soaked toothpick to gently transfer 15 starved third-instar larvae into the Petri dish containing rosette leaves.
      Note: This step needs to be careful and gentle.
    4. Seal the Petri dishes with Micropore tape, and put them in the growth chamber (22 °C, 10-h light/14-h dark).
    5. Feed the larvae for 3 days (Figure 4B), and replace the leaves in each Petri dish by fresh leaves every day.
      Note: Add fresh leaves in time to ensure that the larvae have enough food. The feeding days can be adjusted according to the experimental conditions.
      As the plant hormone jasmonate (JA) plays a vital role in regulating plant defense response against herbivore attack, we conducted the insect feeding assays using the wild type (WT) plants and the coi1-2 mutant, which harbors a point mutation of the JA receptor gene CORONATINE INSENSITIVE1 (COI1) (Xu et al., 2002), and compared their resistance to the S. exigua larvae (Figures 4C and 4D).

  4. Collection of experimental results
    1. After three days of feeding, transfer the larvae into a new Petri dish, and weigh all Spodoptera exigua larvae from each Petri dish.
    2. Place a cotton ball soaked in ether in the Petri dish, and seal the dish with parafilm.
    3. When the larvae are unconscious, use a small pair of tweezers to gently place them on a black cloth, arrange them from large to small, and take photos (Figure 4C).
      Note: Steps D1 and D3 should be gentle to prevent the larvae from being injured and spit out green oral secretions.


      Figure 4. Insect feeding assays with Spodoptera exigua larvae. A. Mature rosette leaves were cut and placed in the plastic Petri dish (150 mm) containing 0.8% agar. B. The leaves after feeding by Spodoptera exigua larvae for 1 day. C. Spodoptera exigua larvae after feeding on rosette leaves of WT and coi1-2 plants for 3 days. Scale Bar = 1 cm. D. Average weight of the larvae fed with rosette leaves of WT and coi1-2 plants for 3 days. Data represent means ± SD (n = 3). Asterisks indicate significant differences from the WT according to Student’s t-test at **, P < 0.01.

Data analysis

Count the weight of each larva in each dish, and calculate the average. Statistical analysis should be done by calculating the average of three independent experiments and standard deviation using Microsoft Excel or any other statistical analysis software. Significance of the difference between two samples can be obtained by performing the Student’s t-test (Figure 4D). Usually, the larvae grow well after eating the leaves. In some cases, one or two larvae will die, and avoid using the dead larvae for analysis. Otherwise, collect all larvae for data analysis.

Recipes

  1. ½ MS medium
    2.215 g Murashige & Skoog basal medium with vitamins
    10 g sucrose
    8 g Bacto-agar
    Add ddH2O to 1,000 ml
    Use KOH to adjust pH to 5.8
    Autoclave at 15 psi, 121 °C for 15 min
  2. 0.8% agar medium
    8 g Agar
    Add ddH2O to 1,000 ml
    Autoclave at 121 °C for 15 min

Acknowledgments

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDB11030200), the Ministry of Science and Technology of China (Grant 2015CB942900), and the National Natural Science Foundation of China (Grants 31730010 and 31770303). This protocol was adapted from the methods described in You et al. (2019).

Competing interests

The authors declare that they have no competing interests.

References

  1. Howe, G. A. and Jander, G. (2008). Plant immunity to insect herbivores. Annu Rev Plant Biol 59: 41-66.
  2. Hu, P., Zhou, W., Cheng, Z., Fan, M., Wang, L. and Xie, D. (2013). JAV1 controls jasmonate-regulated plant defense. Mol Cell 50(4): 504-515.
  3. Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D. and Xie, D. (2002). The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell 14(8): 1919-1935.
  4. You, Y., Zhai, Q., An, C. and Li, C. (2019). LEUNIG_HOMOLOG mediates MYC2-dependent transcriptional activation in cooperation with the coactivators HAC1 and MED25. Plant Cell 31(9): 2187-2205.

简介

[摘要 ] P 兰特-insect互动是研究植物免疫力的一个重要领域。甜菜夜蛾 甜菜夜蛾,是最好的-K的一个nown农业害虫和是通常用来研究植物的相互作用与咀嚼昆虫。这里,我们描述一个协议用于我NSECT ˚F eeding 一个ssays用甜菜夜蛾lavae使用模型宿主植物拟南芥,这是简单且容易进行,并且可被用于评价效果的昆虫生长的宿主基因,从而研究植物对咀嚼昆虫的抵抗力。

[背景 ] 植物面临的各种生物逆境终其一生,如食草动物攻击的d。P个athogen感染。甜菜夜蛾,甜菜夜蛾,是一个世界性的植食性害虫具有广泛的宿主范围,damag 荷兰国际集团各种蔬菜作物和CAU的荷兰国际集团巨大的经济农业损失 (豪和JANDER 2008 ; 胡等人。,20 13 )。甜菜夜蛾升arvae 通常养活两个叶子和果实,并且是极大的破坏性。拟南芥是的寄主植物甜菜夜蛾,而且也是一个经典模式植物,研究植物对食草动物。在这里,我们描述的方法改编自我们以前的工作(你等,2019)笔Ø 进行的˚F eeding 一个与ssays 甜菜夜蛾幼虫对拟南芥莲座叶。通过计算不同基因型的叶片喂食后幼虫的重量,我们能够在实验室环境中评估植物对草食动物攻击的抗性。

关键字:植物-昆虫相互作用, 咀嚼式昆虫, 甜菜夜蛾, 拟南芥, 昆虫喂食实验

材料和试剂


 


正方形皮氏培养皿(100 毫米×100mm的,北京- [R uiaizhengte 乙iological Ť 童占梅Ç O操作。,大号TD 。目录号:YC-HC99050 )
培养皿(150 mm ,康宁,目录号430599 )
1.5毫升微管(AXYGEN,目录号:MCT-150-C)
移液器吸头(AXYGEN,目录号:T-200-Y和T-1000-B)
微孔胶带(3M,Micropore TM ,目录号:1530C-0)
封口膜(Bemis,目录号:PM-996)
Toothp ICK (Suncha,目录号:YQ125 0 )
黑布(北阳,货号:13000133 )
拟南芥
斜纹夜蛾(Keyun)(https://item.taobao.com/item.htm?spm=a1z09.2.0.0.10672e8dnlXuwi&id=567208183626&_u=pmk8luh1123)
人工饲料(我ngredients :小麦胚芽,酵母,角叉菜胶,魔芋粉,山梨酸,维生素C,玉米油,和亚油酸;克运)
(https://item.taobao.com/item.htm?spm=a1z09.2.0.0.10672e8dnlXuwi&id=43498077050&_u=pmk8luh74e5)


Murashige&Skoog含维生素的基础培养基(Phyto Technology Laboratories,目录号:M519)
细菌琼脂(BD,Bacto TM ,货号: 214010)
10%漂白剂(KAO,600米升)
1 M KOH溶液 (甲laddin,目录号:P112281 )
蔗糖(国药集团化学试剂有限公司,公司,。目录号:10021418)
乙醚(国药化学试剂有限公司,目录号:100 09328 )
无菌蒸馏水
营养土壤(苔藓泥炭:ver 石= 2:1,PINDSTRUP,类型:0-10 mm )
½MS 介质(请参见食谱)
0.8%琼脂培养基(请参见食谱)
 


设备


 


移液器(吉尔森的Pipetman ® G)
量筒
[R eagent瓶
冰箱或冷藏室
Ť weezers
植物生长室
方锅
高压釜
层流罩
平衡
佳能相机
统治者
 


 


软件


 


微软Excel
 


程序


 


拟南芥植物的栽培
1. 准备½ MS 固体介质,并且p 提供了足够的介质成正方形P ETRI 菜 ES (100 毫米× 100mm)上,以覆盖约一半的所述深度培养皿(40米升)。      


2. 表面除菌拟南芥种子中1.5米升微管通过在浸泡10 %的漂白剂为15 分钟,T 他N r个通过用漂洗五次EMOVE所有漂白残余物无菌蒸馏水中。      


3. 母猪在种子正方形培养皿含有ES ½ MS 介质(图1A),和s EAL 碗碟带麦克风ropore胶带。      


4. 将培养皿在4 °C 的黑暗环境中放置2 天,以实现高效同步发芽。      


5. 转让菜向生长室在设定22 ℃下用一个10小时的光/ 14小时黑暗光周期为10 一天秒。      


6. Ť ransplant 的苗成方形盆˚F illed与营养土壤(图1B),并保持它们在所述生长室中(22 ℃下,10小时光照/ 14小时黑暗)另外4周(图1C)。      


注意:注意不要损坏幼木的根部。照顾好植物,以防它们受到草食动物或病原体的影响。


 


D:\ Reformatting \ 2020-1-6 \ 1902815-- 1291 Chuanyou Li 810740 \ Figs jpg \图1.jpg


图1. 牛逼他ç ultivation 的拟南芥植物。A. 小号terilize d 拟南芥种子被播种上的正方形培养皿含有½ MS培养基。B.十日龄拟南芥幼苗是牛逼ransplant 编入充满锅营养土壤。C. 拟南芥幼苗盆栽4周长大的用于我NSECT ˚F eeding 一个SSAY 秒。


 


Spodop tera exigua 幼虫的制备
将Spodoptera exigua 鸡蛋放在方形的Pétri 盘中,并用Micropore胶带密封盘(图2A)。
放置在培养皿2 7 ℃的培养箱中以40-50%的相对湿度下孵化(约2 - 3天)。
卵孵化后,立即添加对菜进行日常饮食(图2B),并继续孵育5天。
注意:C UT 的一个rtificial饮食成小块的1 - 1.5 厘米见方,并放置他们在该陪替氏培养皿在从卵或幼虫以一定的间隔。通常,在喂食5天后,幼虫变成三龄幼虫。


 


D:\ Reformatting \ 2020-1-6 \ 1902815-- 1291 Chuanyou Li 810740 \ Figs jpg \ Figure 2.jpg


图2. 牛逼,他孵化的甜菜夜蛾卵。A. 斜纹夜蛾卵。乙。新孵化的幼虫和人工饮食。


 


用牙签吨转让(BOT)的幼虫Ë 在到一个新的Petri 培养皿(视频1和图3A)和饿死他们12小时在实验中使用之前(图3B) 。
注意:此步骤需要小心轻柔。通常情况下,我们ü 知的水浸泡过的牙签升等的幼虫Ë 捕捉并轻轻地转移幼虫,以防止他们受伤。


 


 


D:\ Reformatting \ 2020-1-6 \ 1902815-- 1291 Chuanyou Li 810740 \ Figs jpg \图3.jpg


图3.饥饿处理的甜菜夜蛾幼虫。A.幼虫抓着牙签。B.将T HIRD - 龄幼虫Ë 饥饿d 12小时。


 






视频1. 如何用牙签捕捉幼虫?


 


在教派˚F eeding 一个ssays
1. 准备并把我们足够的0.8%琼脂倒入塑料中       培养皿(150 mm)大约覆盖培养皿深度的一半。


2. 从在土壤中生长的拟南芥植物切下类似大小的成熟莲座丛叶,历时4周,然后将它们放入盛有0.8%琼脂的塑料陪替氏培养皿(150毫米)中(图4A )。      


注意:如果不同基因型的叶子的形状和大小相似,我们通常以图4A所示的方式排列它们。而且,如果叶子的形状和大小相差很大,则需要每次称重叶子,以确保添加相同重量的叶子。


3. 使用一块浸透水的牙签至g ently 转移15个饥饿第三- 龄幼虫Ë 到所述培养皿含有莲座叶。      


注意:此步骤需要小心轻柔。


4。       小号EAL 的培养皿ES 用微孔带,并把它们在生长室(22 ℃下,10小时光照/ 14小时黑暗)。


5. 喂食幼虫3天(图4B ),并每天用新鲜叶子代替每个培养皿中的叶子。      


注:在时间添加鲜叶,以保证幼虫公顷已经足够的食物。在F eeding 天可以根据需要调节的实验条件。


由于植物激素Ĵ asmonate(JA)中扮演一个重要的调节对草食动物攻击植物防御反应的作用,我们进行了昆虫饲养试验使用野生型(WT)植物和coi1-2 MUT 蚂蚁,其港口的点突变该JA受体基因晕斑INSENSITIVE1 (COI1 )(Xu等人,2002) ,并比较它们的电阻的甜菜夜蛾幼虫(图小号4C 和4D )。


 


科尔的实验结果挠度
1. 经过三天内进行付运的馈送,转移幼虫到一个新的培养皿中,并称重所有甜菜夜蛾从每个培养皿幼虫。      


2. 将一个棉球浸泡在乙醚中的培养皿,并且s EAL 培养皿用封口膜。      


3. 当larv 一个ë 是无意识的,使用一个小的对镊子至g ently 将它们放置在黑色布料,arrang È 他们从大至小,并采取照片(图4C) 。      


注:S TEP 小号d 1和d 3 要轻柔,防止幼虫被受伤,吐出绿色的口腔分泌物。


 


 


D:\ Reformatting \ 2020-1-6 \ 1902815-- 1291 Chuanyou Li 810740 \ Figs jpg \图4.jpg


图4. 昆虫˚F EEDI 纳克一个ssays用甜菜夜蛾幼虫。A.将成熟的莲座丛叶切下并置于含有0.8%琼脂的塑料陪替氏培养皿(150mm)中。B. 由斜纹夜蛾幼虫取食1天后的叶子。C. Spodoptera exigua 幼虫取食WT和coi1-2 植物的莲座叶3天后。比例尺= 1厘米。D. 用WT和coi1-2 植物的莲座叶喂食的幼虫的平均体重3天。数据代表平均值± SD(n = 3 )。星号指示从显著差异WT 根据学生吨- 在**,试验P <0.0 1。


 


数据分析


 


计算每道菜中每个幼虫的重量,并计算平均值。统计分析应通过计算平均值来完成的3 独立实验小号和S ^ 使用Microsoft Excel TANDARD偏差或任何其他统计分析软件。Signific 的differenc的ANCE ë 两个样本之间可以通过执行学生获得吨-test (图4 d )。通常,幼虫ē 吃树叶后生长良好。在某些情况下,一个或两个幼虫ê 会死,并避免使用死幼虫Ë 进行分析。否则,收集所有幼虫进行数据分析。


 


菜谱


 


½MS 培养基
2.215 g含维生素的Murashige&Skoog基础培养基


10克蔗糖


8 克琼脂


加入ddH 2 O至1,000 ml


用KOH 调节pH值至5.8


在15 psi ,121 °C下高压灭菌15分钟


0.8%琼脂培养基
8克甲GAR


加入ddH 2 O至1,000 ml


在121 °C下高压灭菌15分钟


 


致谢


 


这项工作得到了中国科学院的战略重点研究计划(Grant XDB11030200),中国科学技术部(Grant 2015CB942900)和国家自然科学基金(Grants 31730010和31770303)的支持。该协议被改编˚F ROM 在你所描述的方法等。(2019)。


 


利益争夺


 


作者宣称他们没有竞争利益。


 


参考文献


 


豪,GA和詹德,G。(2008)。植物对昆虫食草动物的免疫力。植物生物学年鉴59 :41-66。
Hu,P.,Zhou,W.,Cheng,Z.,Fan,M.,Wang,L.和Xie D.(2013)。JAV1 控制茉莉监管的计划牛逼的防守。摩尔电池50 (4 ):504-515。
Xu,L.,Liu,F.,Lechner,E.,Genschik,P.,Crosby,WL,Ma,H.,Peng,W.,Huang,D. and Xie,D.(2002)。SCF COI1 泛素-连接酶复合物是拟南芥茉莉酸酯响应所必需的。植物细胞14 (8 ):1919-1935。
You,Y.,Zhai,Q.,An,C. and Li,C.(2019年)。LEUNIG_HOMOLOG 米ediates MYC2- d ependent 吨ranscriptional 一个ctivation在ç 与ooperation Ç oactivators HAC1和MED25。植物细胞31 (9 ):2187-2205。
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引用:You, Y., An, C. and Li, C. (2020). Insect Feeding Assays with Spodoptera exigua on Arabidopsis thaliana. Bio-protocol 10(5): e3538. DOI: 10.21769/BioProtoc.3538.
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