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Quantification of Serotonin in Rice and Insect Pest and its Functional Analysis in Insects Using Artificial Diet Feeding

水稻及其害虫中血清素含量的测定并通过人工饲养的方式分析血清素在害虫中的作用

LW Long Wang
XG Xiaohao Guo
WH Wenqian Huang
HL Haiping Lu
QS Qing-yao Shu

发布: 2019年02月20日第9卷第4期 DOI: 10.21769/BioProtoc.3173 浏览次数: 5800

评审: Rainer MelzerHsuan ChouAnonymous reviewer(s)

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Abstract

Rice is one of the world’s most important crops, but its production suffers from insect pests. Rice brown planthopper (BPH; Nilaparvata lugens Stål) and striped stem borer (SSB, Chilo suppressalis Walker) are the two most serious pests in rice production. We reported that serotonin is an essential mediator in the interaction between rice and insect. Here, we established a method for extraction and determination of serotonin in rice and BPH.

Keywords: Serotonin (血清素) search Rice (水稻) search Artificial diet (人工饲料) search BPH (BPH) search SSB (SSB) search Insect resistance (抗虫性) search

Background

Serotonin (5-hydroxytryptamine) is an important signaling molecule in animals. It has also been shown to be distributed widely in plants. In our previous research, we reported that deficiency of serotonin in rice plants enhances its BPH and SSB resistance, and the addition of serotonin in plant growth medium results in a loss of insect resistance in mutants (Lu et al., 2018). Here, we provide detailed protocols for determination of serotonin in rice plants, and in SSB and BPH insects, together with the methods for feeding SSB and BPH in artificial diets (added with exogenous serotonin).

Materials and Reagents

  1. Serotonin measurement
    1. Sep-Pak C18 cartridge (Waters, catalog number: WAT020515)
    2. 0.20 µm filter (Millex-LG Millipore, catalog number: SLLGH13NL)
    3. Syringe (10 ml, Aladdin, A2292-05)
    4. Syringe (1 ml, Aladdin, A2292-01)
    5. 50 ml tubes
    6. 2 ml centrifuge tubes
    7. 1.5 ml centrifuge tubes
    8. Rice (Oryza sativa L.) plants, grown in indoor facilities or in the field
      Note: In this experiment, the wild type (WT) cultivar Jiazhe B and its OsT5H (encoding Tryptamine 5 hydrolyase) mutant line Jiazhe LM (Lu et al., 2016) were used.
    9. Brown planthopper (BPH; Nilaparvata lugens Stål), raised in laboratory by feeding on one-month-old TN1 (a susceptible rice variety) plants (originally collected from the field)
    10. Striped stem borer (SSB, Chilo suppressalis Walker), raised in laboratory by feeding on artificial diet
    11. Liquid Nitrogen
    12. Methanol (Aladdin, catalog number: M117118)
    13. Trifluoroacetic acid (Aladdin, catalog number: T103297)
    14. Perchloric acid (Aladdin, catalog number: P112070)
    15. Serotonin standard (Sigma, catalog number:14927)
    16. Ammonium acetate
    17. Formic acid
    18. Nitrogen
    19. Argon

  2. Assay of BPH survival rate by artificial diet feeding 
    1. Glass cylinder (Figures 2A-2B), home-made, ø, 4 cm; height, 8 cm; with 48 small holes (ø, 0.8 mm)
    2. Parafilm M (Parafilm, PM996)
    3. 1.5 ml tubes (centrifuge tubes)
    4. Filter paper (Sangon Biotech, catalog number: F503316)
    5. Plastic tube (24 x 95 mm)
    6. Glycine (Sangon Biotech, catalog number: A100167)
    7. L-Alanine (Sangon Biotech, catalog number: A600022)
    8. L-Arginine hydrochloride (Sangon Biotech, catalog number: A600205)
    9. L-Asparagine (Sangon Biotech, catalog number: A694341)
    10. L-Aspartic acid (Sangon Biotech, catalog number: A600091)
    11. L-Cysteine (Sangon Biotech, catalog number: A600132)
    12. γ-Amino butyric acid (Sangon Biotech, catalog number: A600040)
    13. L-Glutamic acid (Sangon Biotech, catalog number: A600221)
    14. L-Glutamine (Sangon Biotech, catalog number: A100374)
    15. L-Histidine (Sangon Biotech, catalog number: A604351)
    16. L-Methionine (Sangon Biotech, catalog number: A100801)
    17. L-Isoleucine (Sangon Biotech, catalog number: A100803)
    18. L-Leucine (Sangon Biotech, catalog number: A100811)
    19. L-Proline (Sangon Biotech, catalog number: A600923)
    20. L-Lysine hydrochloride (Sangon Biotech, catalog number: A110437)
    21. L-Phenylalanine (Sangon Biotech, catalog number: A600991)
    22. L-Serine (Sangon Biotech, catalog number: A601479)
    23. L-Threonine (Sangon Biotech, catalog number: A610919)
    24. L-Tryptophan (Sangon Biotech, catalog number: A601911)
    25. L-Tyrosine (Sangon Biotech, catalog number: A601932)
    26. L-Valine (Sangon Biotech, catalog number: A600172)
    27. Biotin (Sangon Biotech, catalog number: A100340)
    28. Calcium pantothenate (Sangon Biotech, catalog number: A600683)
    29. Choline chloride (Sangon Biotech, catalog number: A600299)
    30. Folic acid (Sangon Biotech, catalog number: A610466)
    31. Inositol (Sangon Biotech, catalog number: A600536)
    32. Nicotinic acid (Sangon Biotech, catalog number: A610660)
    33. Pyridoxine hydrochloride (Sangon Biotech, catalog number: A600797)
    34. Riboflavin (Sangon Biotech, catalog number: A600470)
    35. Thiamine hydrochloride (Sangon Biotech, catalog number: A500986)
    36. Ascorbic acid (Sangon Biotech, catalog number: A100143)
    37. CaCl2•2H2O (Sangon Biotech, catalog number: A100556)
    38. CuCl2•2H2O (Sangon Biotech, catalog number: A603090)
    39. FeCl3•6H2O (Sangon Biotech, catalog number: A600201)
    40. MnCl2•4H2O (Sangon Biotech, catalog number: A500331)
    41. ZnCl2 (Sangon Biotech, catalog number: A501003)
    42. MgCl2•6H2O (Sangon Biotech, catalog number: A100288)
    43. KH2PO4 (Sangon Biotech, catalog number: A100781)
    44. Sucrose (Sangon Biotech, catalog number: A100335)
    45. BPH artificial diet (D-97) (see Recipes)

  3. Assay of SSB body weight by artificial diet feeding
    1. Plastic barrel
    2. Fresh wild rice (Zizania aquatica) stem (From supermarket) (Han et al., 2012)
    3. Soybean flour (From supermarket, a China brand)
    4. Yeast extract powder (From supermarket, a China brand)
    5. Casein (Sigma, catalog number: C7078)
    6. Sucrose (Sinopharm Chemical Reagent Co., Ltd., catalog number: A100335)
    7. Ascorbic acid (Sangon Biotech, catalog number: A100143)
    8. Sorbic acid (Sangon Biotech, catalog number: SB0899)
    9. Cholesterol (Sangon Biotech, catalog number: C0433)
    10. Choline chloride (AMRESCO, catalog number: 0448)
    11. Wesson's salt Mixture (Sigma, catalog number: W1374)
    12. 40% Formaldehyde (Sinopharm Chemical Reagent Co., Ltd., Catalog number: 10010018)
    13. Methyl parahydroxybenzoats
    14. Agar (Sinopharm Chemical Reagent Co., Ltd., 10000561)
    15. Nicotinic acid amide (Sangon Biotech, catalog number: A510659)
    16. Pyridoxine hydrochloride (Sangon Biotech, catalog number: A600797)
    17. Riboflavin (Sangon Biotech, catalog number: A600470)
    18. Thiamine hydrochloride (Sangon Biotech, catalog number: A500986)
    19. Cyanocoblamine (Fluka, catalog number: 82897)
    20. Folic acid (Sangon Biotech, catalog number: A610466)
    21. Calcium pantothenate (Sangon Biotech, catalog number: A600683)
    22. Biotin (Sangon Biotech, catalog number: A100340)
    23. Semi-artificial diets recipe for SSB (see Recipes)

Equipment

  1. Refrigerated shaker (Hualida Co., Ltd., model: HZ-9210K)
  2. Centrifuge (Eppendorf, model: 5804R)
  3. Rotary evaporator (Shanghai Yarong Biochemical instrument Co., Ltd., model: RE52CS)
  4. -20 °C freezer
  5. High performance liquid chromatography, Agilent 1200 infinity series with diode array detector (DAD) in plant serotonin measurement 
  6. HPLC-MS system, Agilent Technologies 1290 with Agilent 6460 triple mass selective detector under multi reaction monitoring (MRM) working mode in BPH serotonin measurement 
  7. C18 column (Agilent, TC-C18(2), 4.6 x 250 mm, 5 μm) for HPLC
  8. C18 column (Agilent, Zorbax XDB, 2.1 x 150 mm, 3.5 μm) for HPLC-MS
  9. Analytical balance with 0.00001 g accuracy
  10. -80 °C freezer
  11. 4 °C refrigerator
  12. Growth chamber
  13. Blender
  14. Autoclave
  15. Crisper

Software

  1. Variance (ANOVA) program StaView, used for statistical analyses

Procedure

English
中文翻译

文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wang, L., Guo, X., Huang, W., Lu, H. and Shu, Q. (2019). Quantification of Serotonin in Rice and Insect Pest and its Functional Analysis in Insects Using Artificial Diet Feeding. Bio-protocol 9(4): e3173. DOI: 10.21769/BioProtoc.3173.

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分类

植物科学 > 植物免疫 > 植物-昆虫互作

生物化学 > 其它化合物 > 血清素

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