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Jan 2018

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Arabidopsis-Green Peach Aphid Interaction: Rearing the Insect, No-choice and Fecundity Assays, and Electrical Penetration Graph Technique to Study Insect Feeding Behavior
拟南芥与绿桃蚜相互作用:昆虫喂养、无选择性育性分析和昆虫刺吸电位技术在研究昆虫摄食行为中的应用   

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Abstract

Aphids constitute a large group of Hemipterans that use their slender stylets to tap into the sieve elements of plants from which they consume copious amounts of phloem sap, thus depriving the plant of photoassimilates. Some aphids also transmit viral diseases of plants. Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is a polyphagous insect with a host range that covers 50 plant families, is considered amongst the top 3 insect pest of plants. The interaction between Arabidopsis thaliana and the GPA is utilized as a model pathosystem to study plant-aphid interaction. Here we describe the protocol used in our laboratories for rearing the GPA, and no-choice and fecundity bioassays to study GPA performance on Arabidopsis. In addition, we describe the procedure for the electrical penetration graph (EPG) technique to monitor feeding behavior of the GPA on Arabidopsis.

Keywords: Myzus persicae (绿桃蚜), EPG (EPG), Aphid feeding behavior (蚜虫的摄食行为), Plant-aphid interaction (植物 - 蚜虫互作), Sieve element phase (筛管)

Background

Aphids are important pests of plants that utilize their mouthparts, which are modified into stylets, to remove phloem sap from the sieve elements. As part of their feeding process, aphids deposit saliva into the plant tissue. While some salivary components elicit plant defenses, others manipulate host physiology to benefit the insect, including suppressing plant defenses (Nalam et al., 2018). Plants utilize a variety of defenses to control aphid infestation. These include antibiosis, which adversely impacts aphid growth, development and fecundity, and antixenosis, which affects insect behavior, including feeding behavior. These defenses are exerted at various steps, including at the cell surface, during the intercellular penetration of leaf tissue by the insect stylet, when the stylet tip is inside plant cells, and when the stylet tip is in the sieve elements (Nalam et al., 2018). The green peach aphid (GPA), Myzus persicae Sülzer, is an important pest of plants in numerous families, including the Brassicaceae, Solanaceae, Cucurbitaceae, Rosaceae, Asteraceae, Malvaceae, Amaranthaceae (Blackman and Eastop, 2000). In addition, the GPA transmits several viral diseases (Kennedy et al., 1963; Matthews, 1991). During the last decade, the interaction between Arabidopsis thaliana, which belongs to the Brassicaceae family, and the GPA has been increasingly utilized to study plant-aphid interaction (Louis et al., 2012; Louis and Shah, 2013). This pathosystem has facilitated understanding of the physiological and molecular processes that determine the outcome of plant-aphid interaction, including plant defense mechanisms and their impact on insect population growth, fecundity and behavior (Louis et al. 2012; Louis and Shah, 2013).

The direct current (DC)-electrical penetration graph (EPG) system, which measures the electromotive force (EMF) signal and fluctuations in electrical resistance resulting from aphid stylet penetrations, provides a sensitive method to monitor aphid feeding behavior on plants (Tjallingii, 1985; Salvador-Recatalà and Tjallingii, 2015). When the aphid stylet is inserted intercellularly, the voltage is positive and when inserted intracellularly, the voltage is negative (Tjallingii, 2006). The different EPG waveform patterns are indicative of the different activities in which the insect is engaged. Moreover, the duration of each type of waveform provides a quantitative measure of the effect of plant genotype and/or treatment on insect feeding behavior, including the time spent by the insect feeding from the sieve elements. However, despite the success of the Arabidopsis-GPA pathosystem, the protocols utilized to study aphid population growth, fecundity and feeding behavior have not been described in detail. Here, we detail the protocols for rearing a Brassicaceae-adapted colony of the GPA, no-choice assays for monitoring GPA population growth, fecundity assays to monitor insect reproductive rate, and the EPG analysis to monitor GPA feeding behavior on Arabidopsis.


Part I: Rearing the green peach aphid

The green peach aphid (Myzus persicae) colony is maintained on a mix of radish (Raphanus sativus ‘Early Scarlet Globe’) and mustard (Brassica juncea ‘Florida Broadleaf’) plants, which like Arabidopsis belong to the Brassicaceae family. On Brassicaceae, GPA reproduces asexually by releasing live apterous (wingless) nymphs.

Materials and Reagents

  1. T.O. Plastics Standard Flats 1020 tray with bottom holes (Hummert International, model: STE-1020-OPEN - WITH HOLES, catalog number: 11-3000-1 )
  2. T.O. Plastics Standard Flats 1020 tray without holes (Hummert International, model: STE-1020-NH - NO HOLES, catalog number: 11-3050-1 )
  3. Square injection molded plastic pots (4.5” [11.43 cm] width x 3.75” [9.53 cm] height) with holes at the bottom (International Greenhouse, catalog number: CN-SQK )
  4. Twist ties
  5. Biohazard autoclave bags (Fisher Scientific, catalog number: 01-830D )
  6. NalgeneTM polypropylene heavy duty sterilizing tray (Thermo Fisher Scientific, catalog number: 6900-0020 )
  7. Soil Mix (Sunshine® Mix #8, Sun Gro Horticulture, model: Fafard®-2 )
  8. Radish seeds (Radish Early Scarlet Globe) (Main Street Seed & Supply, catalog number: 13307-13 )
  9. Mustard seeds (Florida Mustard Broad Leaf) (Main Street Seed & Supply, catalog number: 12501-13 )
  10. Green peach aphid colony (Specimen number 194 deposited with Kansas State University Museum of Entomological and Prairie Arthropod Research)

Equipment

  1. Plant growth chamber (Percival Scientific, model: AR-66L2 )
    Note: Programmed for a 14/10 h day (80-100 μE m-2 sec-1)/night photoperiod at 22 °C.
  2. Autoclave

Procedure

  1. Place soil in an autoclave bag. Break up any lumps and add tap water. Knead the soil-water mix, till the soil is evenly moist. Close the bag with a plastic tie. With a scissor make four small incisions in the plastic bag to facilitate venting after the bag is removed from the autoclave at the end of Step 2. 
  2. Place the bag containing soil in an autoclavable plastic tray and autoclave for 1 h at 121 °C/15 psi on a liquid cycle. After the autoclaving is completed, pull the tray with soil out of the autoclave and let the soil cool down to room temperature. It is best to let the soil cool down overnight.
  3. To prepare pots for planting radish and mustard, take a flat plastic tray with holes and place it inside a flat plastic tray without holes. Place eight, 4.5” (11.43 cm) pots in the tray with holes. Fill these pots loosely with the wet autoclaved soil, making sure to fill the pots to the top. 
  4. Sprinkle 30-40 seeds each of radish and mustard on the top of the soil in each pot (see Figure 1).


    Figure 1. Radish and mustard seeds in a 4.5” (11.43 cm) pot

  1. Spray the seeds generously with water.
    Note: All the seeds should be nicely sprayed with water. If seeds are left dry, they will not germinate.
  2. Place the trays with the pots containing the fresh radish and mustard seeds in the growth chamber that contains GPA-colonized mix of radish and mustard plants (Figure 2). The tray with fresh seeds is placed adjacent to the tray containing GPA colonized mustard and radish plants. The growth chamber should be preferably located in a contained area where plants for other use are not cultivated.


    Figure 2. Radish and mustard plants with GPA (indicated by black arrows)

  3. The radish and mustard seed mix should begin germinating within 2-3 days. The aphids will begin moving to the fresh plants in a few days.
  4. Repeat this procedure every week to maintain a healthy GPA colony. 
  5. Unwanted plants and soil are emptied into autoclave bags and autoclaved for 1 h at 121 °C/15 psi on a liquid cycle before disposing of.
    Steps to prevent insect escaping out of contained facility: If feasible, have the insect colony in a room that is physically separated from the room in which plants are normally cultivated. To limit spread of the insect out of the insect room, avoid going from the insect room to a room where plants are cultivated. If feasible, avoid wearing shirts/lab coats with long sleeves, or roll-up the sleeves so that insects do not accidentally crawl on to your shirts and escape out of the insect room. Further, wash hands well before conducting any other work.

Part II: No-choice bioassay

The no-choice bioassay provides a simple assay to compare aphid population growth on plants of different genotypes. It provides a measure of differences between the resistance levels of plants of different genotypes. It can also be used to compare the impact of various treatments (e.g., chemicals) on aphid population growth. The no-choice assay measures the combined effect of antibiosis and antixenosis. Antibiosis is the effect of host defenses on insect growth, development and reproduction, while antixenosis reflects the effect of plant mechanisms on insect behavior. The Electrical penetration graph technique, which is described later, measures the impact of host genotype or treatments on insect feeding behavior.

Materials and Reagents

  1. T.O. Plastics Standard Flats 1020 tray with bottom holes (Hummert International, model: STE-1020-OPEN - WITH HOLES, catalog number: 11-3000-1 )
  2. T.O. Plastics Standard Flats 1020 tray without holes (Hummert International, model: STE-1020-NH - NO HOLES, catalog number: 11-3050-1 )
  3. Pots for rearing green peach aphid: Square injection molded plastic pots (4.5” [11.43 cm] width x 3.75” [9.53 cm] height) with holes at the bottom (International Greenhouse, catalog number: CN-SQK )
  4. Pots for cultivating Arabidopsis: Square injection molded plastic pots (3.5” [8.9 cm] width x 37/8” [9.84 cm] height) with holes at the bottom (International Greenhouse, catalog number: CN-SQK )
  5. Soil Mix (Sunshine® Mix #8, Sun Gro Horticulture, model: Fafard®-2 )
  6. Clear vinyl propagation dome to fit 1020 flats (Hummert International, catalog number: 11-3360-1 )
  7. Twist ties
  8. Biohazard autoclave bags (Fisher Scientific, catalog number: 01-830D )
  9. NalgeneTM polypropylene heavy duty sterilizing tray (Thermo Fisher Scientific, catalog number: 6900-0020 )
  10. Camel hair paint brush (size 2 or smaller) (Fisher Scientific, General Data, catalog number: 15-183-35 )
  11. Tooth picks
  12. Labeling tape 0.5” (1.27 cm) width (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F13481-0050 )
  13. Fine-tip permanent alcohol/waterproof lab marker (VWR, catalog number: 52877-310 )
  14. Green peach aphid colony (Specimen #194 deposited with Kansas State University Museum of Entomological and Prairie Arthropod Research)
  15. Arabidopsis thaliana seeds accession Columbia (However, other accessions should also be fine)
  16. Radish seeds (Radish Early Scarlet Globe) (Main Street Seed & Supply, catalog number: 13307-13 )
  17. Mustard seeds (Florida mustard broadleaf) (Main Street Seed & Supply, catalog number: 12501-13 )
  18. Peters 20:20:20 General Purpose fertilizer (Hummert International, catalog number: 07-5400-1 )

Equipment

  1. Two plant growth chambers (Percival Scientific, model: AR-66L2 )
    Note: The growth chambers are programmed for a 14/10 h day (80-100 μE m-2 sec-1)/night photoperiod at 22 °C. One is required for cultivating Arabidopsis, and the second chamber is required for the no-choice assay with aphids.
  2. Autoclave
  3. Cold room or refrigerator (4-10 °C)

Procedure

  1. Propagating Arabidopsis for experiments with the GPA
  1. Autoclave the soil and prepare the 3.5” (8.9 cm) pots with soil as described above in Part I Procedure. At least five pots are required for each Arabidopsis genotype. Stick a 2-3 cm length of labeling tape on each pot and with a fine-tip waterproof marker, write down the plant genotype.
  2. Place the soil-filled pots in the tray with holes. Each tray can take a maximum of 18, 3.5” (8.9 cm) pots. A minimum of five pots are required for each Arabidopsis genotype.
  3. Dissolve 0.1 g of Peter’s 20:20:20 fertilizer in one liter of tap water.
  4. Fill the tray without holes with the fertilizer solution to a depth of 1-1.25” (2.54-3.81 cm).
  5. Place the tray (with holes) containing the soil-filled pots in the tray containing the fertilizer solution. The fertilizer solution will rise up through the soil by capillary action. When the top of the soil appears moist (approximately 10-15 min), lift the top tray containing the pots and place it at a slight angle so that it sits on the top edges of the lower tray. Allow the excess fertilizer solution to water drain out into the lower tray.
  6. Soil is now ready for sowing seeds. At least 10 Arabidopsis plants (two per pot) are required for each genotype. Place two Arabidopsis seeds of the same genotype diagonally across from each other, 1-1.25 cm from the edge of the pot. The seeds can be picked up, one at a time, by touching the moistened tip of a toothpick to the seed. Seeds will adhere to the tip of the toothpick. Seeds can now be placed on soil by touching the seed, which is adhered to the tip of the toothpick, to the moistened soil surface.
    Note: Do not push the seed into the soil and have only seeds of the same genotype in each pot.
  7. Cover the tray with the clear vinyl propagation dome.
  8. Put the tray in a cold room or refrigerator for 48 h (light is not required during this period). This will allow the seeds to imbibe water and increase the chance of seeds germinating uniformly.
  9. At the end of the cold treatment, move the trays and pots covered with the clear vinyl propagation dome into the growth chamber. The seeds should germinate, and a pair of green cotyledons should emerge within 2-3 days.
  10. Once the first pair of true leaves has appeared (usually 8-9 days after placing the pots in the growth chamber), remove the transparent plastic dome. Leave the plants uncovered in the growth chamber.
  11. Fertilize plants every two weeks, by placing the pots contained in a tray with holes in a tray containing the fertilizer solution (0.1 g Peter’s 20:20:20 fertilizer per liter of water).

  1. The No-choice bioassay setup and analysis
  1. When the plants are 24-26 days old (see Figure 3), take them to the insect room.


    Figure 3. Arabidopsis plants for no-choice bioassay

  2. Use a camel hair paint brush to gently pick up adult apterous (wingless) aphids (~1 mm size), one at a time, from the radish/mustard plants, and place them at the center of each Arabidopsis plant. Repeat the process till 20 aphids have been placed on each Arabidopsis plant. 
  3. Place the infested plants in the growth chamber. Space the pots to ensure that the pots and plants in adjacent pots are not touching each other.
  4. Fouty-eight hours later, count the total number of aphids (nymphs + adults) on each plant. Most of the aphids will be on the abaxial side of the leaves (see Figure 4).


    Figure 4. GPA on the abaxial side of Arabidopsis leaves

  5. When comparing insect population size on two genotypes (see Figure 5), Student’s t-test can be used to determine if the mean of insect population size on the two genotypes is significantly different (P < 0.05).


    Figure 5. Representative no-choice experiment data. No choice experiment was conducted with the wild type and a mutant genotype of Arabidopsis that exhibits lowered resistance to the GPA. Aphid counts (adult + nymphs) were taken 2 days-post-infestation. Shown is the average aphid counts of aphids on each plant (n = 10). Error bars represent standard error. An asterisk indicates that values between the two genotypes are significantly different (P < 0.05; t-test).

  6. At the end of the experiment, the plants and the soil are emptied into autoclave bags and autoclaved for 1 h at 121 °C/15 psi on a liquid cycle before disposing of.

Part III: Fecundity assay

The fecundity assay is used to determine the reproductive capacity of the GPA on an individual genotype or to compare the reproductive capacity of the GPA between plants of different genotypes, or in plants treated with a chemical compared to control plants.

Materials and Reagents

  1. Supplies for propagating GPA and cultivating Arabidopsis and radish/mustard plants as described above in Part I and Part II, respectively
  2. Camel hair paint brush (size 2 or lower) (Fisher Scientific, General Data, catalog number: 15-183-35 )
  3. 14 day old Arabidopsis plants; two per pot
  4. Petri dish 100 mm wide x 15 mm deep (Fisher Scientific, catalog number: FB0875713 )
  5. Radish seeds (Radish Early Scarlet Globe) (Main Street Seed & Supply, catalog number: 13307-13 )
  6. Mustard seeds (Florida mustard broad leaf) (Main Street Seed & Supply, catalog number: 12501-13 )

Equipment

  1. Two plant growth chambers (Percival Scientific, model: AR-66L2 )
    Note: The growth chambers are programmed for a 14/10 h day (80-100 μE m-2 sec-1)/night photoperiod at 22 °C. One is required for cultivating Arabidopsis, and the second chamber is required for the fecundity assay with aphids.
  2. Autoclave
  3. Cold room or refrigerator (4-10 °C)

Procedure

  1. Prepare soil and plant Arabidopsis and radish/mustard seeds as described above in Part I and Part II Materials and Reagents. 
  2. A day before initiating experiment with Arabidopsis, with a camel hair paint brush place several apterous adult (~1-1.5 mm) insects on healthy radish/mustard plants. 
  3. Next day, with a camel hair paint brush, collect the newly emerged nymphs in a Petri dish.
  4. Use the paint brush to release two 1-day-old nymphs on each Arabidopsis plant. Each nymph is released on a separate leaf. At least 10 Arabidopsis plants are required for each genotype.
  5. Place the infested plants in the growth chamber. Most of these nymphs will grow in size and start reproducing in approximately 6-8 days. The fecundity assay will determine the number of nymphs produced by these (mother) aphids.
  6. From Day 4 onwards, check each plant every 2 days for newly emerged nymphs. Count the number of nymphs and discard them, leaving only the mother aphids on the plant.
  7. Continue counting the number of newly emerged nymphs for a period of 17-18 days post release of the mother aphid on the Arabidopsis plant.
  8. Determine the total number of nymphs that were recovered from each plant over the duration of the experiment. Fecundity, which is represented as the average number of nymphs released per day, per aphid, is calculated with the following equation:

    Fecundity = N ÷ 2(D)

    where, N is the total number of newly emerged nymphs recovered from each plant over the duration of the experiment and ‘D’ is the total number of days in the experiment. The number 2 indicates the number of insects that were released on each Arabidopsis plant at the start of the experiment.
  9. When comparing GPA fecundity on two genotypes (Figure 6), Student’s t-test can be used to determine if the fecundity value on the two genotypes is significantly different (P < 0.05) from each other.


    Figure 6. Aphid fecundity assay. Shown is the average number of nymphs released per mother aphid per day over an 18 day period on Arabidopsis wild-type and mutant genotypes (n = 10). Error bars represent standard error. An asterisk, indicates that values between the two genotypes are significantly different (P < 0.05; t-test).

  10. At the end of the experiment, the plants and the soil are emptied into autoclave bags and autoclaved for 1 h at 121 °C/15 psi on a liquid cycle before disposing of.

Part IV: Electrical penetration graph to study the green peach aphid feeding behavior

The Electrical penetration graph (EPG) measures the time spent by the aphid on various feeding activities. It can be used to determine the effect of plant genotype and or environmental factors (e.g., chemical treatment) on aphid feeding behavior. A cartoon of the EPG set-up is shown in Figure 7. In EPG, an uninsulated electrode, the plant electrode, is placed in the soil in which a plant is growing, thus electrifying the plant with a very low-voltage, low amperage current. An insect electrode, which at one end contains an extremely fine and flexible gold-wire is glued to the dorsum (back) of an aphid. The other end of the gold wire is connected to a copper wire, which in turn is connected to a brass nail that is hooked up to an amplifier. When the aphid is walking on the surface of the plant tethered to gold wire of the insect electrode, the 'switch' is open in this electrical circuit. However, as the stylet contacts the conductive tissues of the electrified plant, a potential drop occurs and unique waveform patterns reflecting different activities are produced, including (i) non-probing phase (baseline), (ii) pathway phase when stylet is inserted into leaf, but not in the phloem, (iii) time to first probe, (iv) sieve element phase (SEP) when the insect is feeding from sieve elements, (v) time to first SEP, and (vi) xylem phase. Below we describe the steps involved in preparation of the insect probe, the attachment of the insect electrode to the aphid, data acquisition and analysis.


Figure 7. Illustration of an EPG setup. A thin gold wire (insect electrode), which permits unhindered movement of aphid, is connected to the EPG monitor. An output wire (stiff copper wire), which is part of the plant electrode, is inserted into the soil of the pot, in which the plant is rooted. The other end of the plant electrode is connected to the EPG monitor. The plant is electrified with a low-voltage, low amperage current. Once the aphid starts feeding on the plant, the aphid stylet comes into contact with the electrified plant, the circuit will be closed and current will flow through the insect and into the monitor, thus producing different waveforms. Illustration by Nick Sloff, taken with permission from Louis et al. (2012) The Arabidopsis Book e0159. doi: 10.1199/tab.0159, www.arabidopsisbook.org).

Materials and Reagents

  1. Supplies for propagating GPA and cultivating Arabidopsis and radish/mustard plants as described above in Part I and Part II, respectively
  2. Thin gold wire Ø 18 μm (thickness range 10-20 μm; available at https://www.epgsystems.eu/epg/products)
  3. Brass connector pins/nails, Ø 1.2 mm (comes with Giga-8d EPG System; http://www.epgsystems.eu/)
  4. Copper wire Ø 0.2 mm (AliExpress, model: YT1303
  5. Adjustable swivel clamps to hold the EPG probes (EPG Systems, comes with Giga-8d EPG System; http://www.epgsystems.eu/)
  6. Lead soldering roll (can be purchased from a local hardware store)
  7. Petri dish 100 mm wide x 15 mm deep (Fisher Scientific, catalog number: FB0875713 )
  8. Camel hair paint brush (size 2 or lower) (Fisher Scientific, General Data, catalog number: 15-183-35 )
  9. Dissecting needle (Thermo Fisher Scientific, catalog number: 19010 )
  10. Paper pins or T pins
  11. Kimwipes® (KCWW, Kimberly-Clark, catalog number: 34120 )
  12. Andwin Scientific Miracloth pore size 22-25 μm (Fisher Scientific, catalog number: NC9147303)
    Manufacturer: Andwin Scientific, catalog number: 475855 .
  13. Styrofoam/Polystyrene box
  14. Radish/mustard plants for propagating GPA
  15. 21-28 days old Arabidopsis plants; one per pot
  16. Conductive silver paint-Colloidal Silver (Ted Pella, catalog numbers: 16031 , 16034 )
    Note: Alternatively, prepare your own silver glue as described at https://www.epgsystems.eu/downloads-install-files-manuals/file/24-add-ons-and-hints

Equipment

  1. Tweezers
  2. GIGA-8 direct current amplifier (http://www.epgsystems.eu/)
  3. EPG probes (http://www.epgsystems.eu/)
  4. Plant electrode (http://www.epgsystems.eu/)
  5. Grounding cable and test cable (see GIGA manual; http://www.epgsystems.eu/)
  6. Stereo dissecting microscope 
  7. A computer to record and store the real-time monitoring of EPG waveforms
  8. Faraday cage to prevent external noises*
  9. Vacuum-operated plate for wiring aphids*
  10. Soldering iron (can be purchased from a local hardware store) to attach copper wire to brass nails
  11. Fume hood for soldering

Note: *Construction of the Faraday cage and the vacuum suction device have been explained in detailed by W.F. Tjallingii in the Giga 8d Manual (https://www.epgsystems.eu/downloads-install-files-manuals/file/24-add-ons-and-hints).

Software

  1. Stylet+ (http://www.epgsystems.eu/)
  2. JKI Macro for Excel (https://www.epgsystems.eu/downloads-install-files-manuals/category/4-epg-data-processing)
  3. Excel Workbook for automatic parameter calculation (Sarria et al., 2009) 
  4. Ebert 1.0 for parameter calculation (http://www.crec.ifas.ufl.edu/extension/epg/sas.shtml)
  5. Microsoft Excel®
  6. Analysis of Variance (ANOVA) (SAS Institute Inc, SAS v5.1)
  7. Minitab® 18.1 for non-parametric Analysis (Kruskal-Wallis Test and Mann-Whitney U test)

Procedure

The following protocol is provided for studies on Arabidopsis thaliana and the green peach aphid (GPA), Myzus persicae.

  1. Preparation of the insect electrode
    The insect electrode (Figure 8) consists of a brass connector pin (Ø 1.2 mm) to which a copper wire (2-4 cm long; Ø 0.2 mm) is attached by soldering the wire to the head of the brass connector pin (Video 1; Part A). A gold wire (2-4 cm long; Ø 18 μm) is attached to the opposite end of the copper wire using silver glue or colloidal silver paint (Video 1; Part B).


    Figure 8. The various components of an insect electrode

    Video 1. Preparation of insect electrode

    1. Using the soldering bolt, melt some soldering wire at its tip onto a hard, heat resistant surface.
    2. Apply soldering fluid to the head of the brass connector pin and dip the brass connector pin into the melted soldering fluid.
    3. Immediately bring the brass connector pin with the soldered fluid on the head into contact with the one end of a 2-4 cm piece of precut copper wire (Video 1; Part A).
    4. Hold the copper wire in place until the soldering fluid cools and solidifies on the brass connector pin-head creating a strong bond with the copper wire.
      Note: The above Steps A1-A4, should be performed under the fume hood.
    5. Dip the free end of the copper wire into a tube containing silver glue or colloidal silver paint in order to create a fine sheath of glue/colloidal paint on the free end.
      Note: In order to ensure a uniform distribution of silver particles and glue, thoroughly shake the sliver glue/colloidal paint before use.
    6. Touch the glue/paint end of the copper wire to the end of a 2-4 cm piece of precut gold wire and gently lift the gold wire. Alternatively, make a few turns of the gold wire on the copper wire and apply the sliver glue/paint over it (Video 1; Part B).
    7. Using a dissection needle, gently manipulate the gold wire to distribute the glue/paint to overlap the copper wire and about 0.5 cm of one end of the gold wire (Video 1; Part B).
    8. Allow the glue/paint to dry completely before applying a second coat of the glue/paint using the tip of the dissection needle to create a strong bond between the copper wire and the gold wire. This process is also done to ensure that there are no glue/paint-free parts of contact between the copper wire and the gold wire.
    9. After the glue/paint is dry, the insect electrode is ready and can be stored in a styrofoam/polystyrene box.
      Note: The insect electrode can be prepared using either a stereo dissection microscope or under sufficient illumination on a laboratory bench.

  2. Preparation of aphids for wiring
    Collect enough apterous adult aphids (at least 10-15 for preparing eight insect electrode-wired insects) from the aphid colony and transfer them to a Petri dish (100 mm wide x 15 mm deep).
    Prior to the wiring, aphids are starved for one hour to ensure feeding upon the start of the EPG experiment.
    Note: A Giga-8 system can at a time, simultaneously monitor the feeding behavior of a maximum of 8 aphids. Hence, to obtain readings from 15-20 aphids, the experiments will have to be conducted on multiple days.

  3. Connecting aphids to the insect electrode
    The insect electrode (Figure 8) described above is connected to the aphid with a silver glue/colloidal paint (Figures 9A and 9B). For the following procedure, the use of a stereo dissecting microscope will greatly ease the process. A vacuum suction device can be utilized to hold the aphid in place while attaching the insect electrode to the dorsum of the aphid. Alternatively, the aphid can be placed on a KimWipe® or MiraCloth® in order to reduce the mobility of the aphid.


    Figure 9. A wired green peach aphid. A. Cartoon depicting the gold wire component of the insect electrode attached to the dorsum of a GPA with sliver paint/glue. B. An image of a GPA connected to the insect electrode via the gold wire and sliver paint/glue.

    1. Ensure that a smooth emulsion of the silver glue/colloidal paint is available by shaking the tube thoroughly.
    2. With a camel hair paint brush lift an aphid from the Petri dish where the apterous adult aphids are being starved.
    3. Using a pin/dissecting needle, apply a small drop of glue/colloidal silver paint on the dorsum of an adult aphid (Figures 9A and 9B). Allow the glue/colloidal silver paint to dry for several minutes. Ensure that the glue/colloidal silver is not applied to the head, antennae or legs of the insect.
    4. Once the first drop of glue/colloidal sliver paint is dry, apply a second drop of glue/colloidal sliver paint to the same spot on the back of the aphid.
    5. Immediately, insert the free end of the gold wire of an insect electrode into the second drop of glue/colloidal silver paint (Video 2). Ensure that the other end of the gold wire points upwards from the insect and does not hinder the movement of the aphid’s legs or antennae.

      Video 2. Connecting an aphid to the insect electrode. Connecting the aphid to the gold wire end of the insect electrode with silver glue/colloidal silver paint.

    6. Hold the insect electrode in place until the glue/colloidal paint is dry. This process will take couple of minutes, during which minimal movement of the aphid and the insect electrode is recommended.
      Note: If the glue/colloidal silver paint smears over the antennae or the legs of the aphid or if the gold wire of the insect electrode does not attach, discard the aphid and repeat the process with a fresh aphid. Avoid adding a third droplet of glue/colloidal paint on the aphid back.
    7. At this point, turn off the suction device (if using one) or lift the aphid away from the stage of the stereo dissecting microscope. The camel hair paint brush can be used to assist in lifting the aphid.
    8. The insect electrode with attached aphid can then be stored until use, by inserting the brass connector pin end into a styrofoam/polystyerene block (Video 3).

      Video 3. A GPA tethered to an insect electrode

    9. Repeat Steps C1-C8 until all the aphids needed for the EPG experiment are prepared and ready.

  4. Plant access
    1. Plants for EPG should be well watered the day before the experiment. Place the Arabidopsis plants in the Faraday cage on a non-conductive surface (Figures 10A and 10B). Examples of non-conductive surfaces include Petri-dishes, cardboard from shipping containers or pieces of styrofoam cut to the appropriate size to be placed under the pots.
    2. If using all 8 channels of the Giga-8, arrange eight Arabidopsis plants in the Faraday cage in a manner that the no part of the leaves or pots touch each other.
    3. Insert the plant electrode into the soil of each pot as close to the base of the pot as possible causing minimal damage to the plant roots.
      Note: The plant electrode (http://www.epgsystems.eu/) consists of a copper wire (10-12 cm long; Ø 0.6 mm) connected to a cable and connector pin for connection to the Giga-8 system.
    4. Insert the brass pin of the insect electrode with a wired aphid into the input connector of the EPG probe. The proper position of the insect should be such that the legs should be in the walking position on the plant surface.
    5. Lift the EPG probe (Figure 10C) so that the insect is not in contact with the plant surface and then proceed to connect the remaining prepared insect electrodes with wired aphids to the remaining channels.
    6. If required, use the swivel clamps to adjust the height of the EPG probes (Figure 10C).
    7. Once all the insect electrodes are connected and the aphids are hanging a few centimeters above the selected leaves/plants, start data acquisition using the Stylet+ software (Detailed methodology of the using the software can be found in the Stylet+ manual at https://www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software).
    8. Lower the insects one at a time on to the plant surface and ensure that the aphid is able to place its legs on the plants surface and that there is sufficient slack to allow the aphid to move around to find an appropriate place for stylet penetration (Figure 10D).


      Figure 10. The EPG setup. A. A Faraday cage containing the Giga-8 setup; B. A close up of the Giga-8 setup monitoring the activity of five GPA on Arabidopsis; C-D. A close-up view of GPA attached by the aphid electrode to the Giga-8 system.
      Note: Some users might want to increase the length of the gold wire in the insect electrode to allow for sufficient room for the aphid to move. However, a longer gold wire can result in entanglements on plant structures resulting in noise during the EPG recording.

    9. The amplifier settings may be adjusted once the aphids have started probing (details can be found in Giga 8d Manual: https://www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software).

  5. Important considerations
    To obtain data with minimal variance between samples, the following considerations should be taken into account.
    1. Optimal EPG Recording time: For GPA feeding on Arabidopsis, the optimal recording time is 8 h. Longer recording times are possible. Pilot experiments are necessary to accurately determine the duration of EPG recordings.
    2. Age of plants: Ensure that the age of the plants is as close as possible. In order to achieve this, follow staggered planting so that plants for the experiments are of uniform age.
    3. Site of aphid feeding: If possible and your EPG set-up allows for it, place the aphids connected to the insect electrode on the same-aged leaf of each plant i.e., avoid placing aphids on leaves of different ages.
    4. Time of day: Aphids exhibit circadian rhythm like any other living organism and aphid feeding behavior may vary with time of day. Avoid setting up and running EPG experiments during the night. Endeavor to run all of your experiments at the same time of the day.
    5. Number of replications: The appropriate number of replicates should be such that they compensate for behavior variability among individual aphids. Twenty successful replicates are ideal, but a minimum of 15 is essential for all experiments.

  6. Analysis of EPG waveforms
    The Stylet+ Analysis (Ana) is used for EPG waveform analysis. Feeding by the GPA on Arabidopsis plants reveals the presence of nine distinct waveforms: A, B, C (Pathway phase), pd (Potential drops), E1 and E2 (Phloem phase or Sieve element phase, SEP), G (Xylem phase), F (Derailed stylet phase) and NP (non-probing) (Figure 11). Further details on the characteristics of the waveforms can be found at: https://www.epgsystems.eu/downloads-install-files-manuals/category/4-epg-data-processing.

    1. Descriptions of Waveforms
      1. During the EPG experiment, periods of inactivity when no stylet penetration (Figure 11) occurs and the aphid stylet is retracted and not located in any host tissue can be observed. This period is referred to as non-probing phase (NP).


        Figure 11. A representative EPG waveform pattern of GPA feeding on Arabidopsis wild-type accession Columbia plant over a 5 h period. The Phloem or sieve element phase (SEP), Xylem Phase (XP); Pathway Phase (PP), and Non-probing phase (NP) are identified.

      2. The waveforms A, B and C (Figure 12A) represent the location of the stylet in the epidermis and mesophyll tissues. The A waveform indicates the presence of the aphid stylet in the epidermis, the B waveform indicates the presence of the stylet in the epidermis or mesophyll and the C waveform represents the presence of the stylets in any tissue. The three waveforms overlap and are hard to tease apart. Therefore, they are lumped together as ‘pathway phase’ or ‘stylet pathway’ and are labeled as waveform C in EPG analysis (Figure 12A). Pathway phase (PP), or stylet pathway phase, is referred to as the phase of stylet penetration that is not the phloem or xylem phase and encompasses a variety of stylet penetration behaviors including intercellular stylet advancement and withdrawal, and brief intracellular punctures by the stylet tips. It is during this probing phase that the insect attempts to locate its primary ingestion site (i.e., the sieve element), and makes decisions regarding host acceptance or rejection.
      3. Potential drops or pd represent the punctures of plant cells by the stylet (Figure 12A). The pd waveform can be further sub-divided into II-1, II-2 and II-3 (Figure 12B). These characterizations are important if the experiment involves studying the inoculation of persistent viruses by the aphids. These waveforms are not required to be labeled if not required by the research question.


        Figure 12. Aphid waveforms of the pathway phase including a potential drop. A. The pathway phase consists of A, B and C waveforms lumped into one category as C. B. A potential drop (pd) showing II with 3 sub-phases (II-1, II-2 and II-3).

      4. SEP occurs when the stylet tips are in a phloem sieve element, which is the site of phloem sap ingestion by aphids. The SEP consists of E1 (Figures 13A and 13B) and E2 (Figures 13A and 13C). During E1, the aphid stylet is located in the sieve elements and the aphid is actively salivating, presumably in an effort to suppress host responses. During E2, the aphid stylet is located within the sieve element and the aphid is ingesting phloem sap. 


        Figure 13. Aphid waveforms of the Sieve element phase (SEP). A. SEP phase showing E1 and E2; B-C. Expanded view of E1 and E2, respectively.

      5. During the G waveform (Figure 14), the aphid stylet is located in the xylem and the aphid is ingesting xylem sap.


        Figure 14. Aphid waveforms of the xylem feeding/ingestion phase (G)

      6. The waveform F refers to derailed stylets (Data not shown), meaning the aphid stylet is encountering penetration difficulties. During this phase, the aphid stylet is located in the host tissue but due to difficulties in stylet mechanics, the aphid is not involved in feeding behaviors.

    2. Waveform analysis
      1. The identification of various waveforms are performed visually and labeling the waveforms is performed using the Stylet+ software (detailed methodology to use the software can be found at: https://www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software).
      2. Stylet+ allows the user to create analysis grids and store the analysis data in “.ana” files that are required for downstream data processing.
      3. If waveform analysis reveals that an aphid spent > 70% of the recording time in Np+F+G activities, discard that aphid.

  7. Calculation of EPG events
    The length of EPG events and the complexity of the parameters used to explore insect feeding behavior make the analysis of EPG data a time-consuming process. The ability to automatically calculate a large number of EPG parameters greatly reduces the time involved, increases efficiency and provides an accurate view of aphid insect probing and feeding behavior. The availability of several published programs: Backus 1.0 (Backus et al., 2007), Sarria Workbook version 4.4.3 (Sarria et al., 2009), EPG-Calc 6.1 (Giordanengo, 2014), Excel macro JKL 2.0 (www.epgsystems.eu) and Ebert 1.0 (Ebert et al., 2015) are valuable for accelerating the speed and accuracy of processing the large amount of data generated during EPG experiments. The choice of the program that researchers choose for data analysis depends on the experimental setup, insect-plant system and accessibility to the software tools. Ebert et al. (2015) provide a comprehensive review of three of the above-listed programs (Backus 1.0, Sarria Workbook version 4.4.3 and Ebert 1.0).

Data analysis

The data obtained from EPG recordings is considered non-parametric data since they do not conform to the assumptions of one-way ANOVA and must therefore be analyzed using appropriate statistical tests or the data can be subjected to transformation to meet the assumptions of ANOVA. Two methods of analysis are appropriate:

  1. Data analysis without transformation: The different parameters obtained from EPG analysis worksheets listed in G are first compared using the Kruskal-Wallis test, which is a distribution-free test. Parameters that show significant differences (α = 0.05) between treatments (if more than two) can be further evaluated using the Mann-Whitney U (MWU) test. MWU is used to compare differences between two treatments and therefore, repeated MWU tests have to be carried out between all treatments to determine significant differences (α = 0.05).
    For example, if your EPG experiment consists of three treatments: T1, T2 & T3, and the Kruskal-Wallis test indicates that there are significant differences between the three treatments for parameter X (P ≤ 0.05), then to identify which treatments are significantly different from each other, the MWU tests are carried out T1 vs. T2, T1 vs. T3 and T2 vs. T3. 
  2. Data analysis with Rank transformation: In order to use ANOVA to analyze the different parameters obtained from the EPG recordings, the data can be rank transformed. Rank transformation means that the values of the activity of every treatment for each parameter are arranged in rank order. In order to carry out this transformation, the data for each parameter for all treatments is arranged in a sequential manner using the RANK.AVG function in MS Excel. This function returns the rank of a number in a list of numbers and if more than one value has the same rank, it returns the average rank. The rank-transformed data for each parameter can then be analyzed using ANOVA (either is SAS or Minitab) to determine differences between treatments. Parameters that show a P-value of ≤ 0.05 can be subjected to a post-hoc test such as Tukey’s Honest Significant Difference test to determine the difference between means of the different treatments.
    Note: Ebert 1.0 is a SAS program that provides both data compilation and statistical analysis of EPG variables. Data analysis is performed using a mixed model analysis of variance (Ebert et al., 2015). 

Notes

  1. Arabidopsis plants should not be overwatered. Overwatering increases chances of algal and fungal growth in soil. It is best to sub-irrigate only when the soil surface shows the first sign of drying. This will also facilitate healthy growth of Arabidopsis.
  2. Maintain a back-up of the GPA colony in a separate growth chamber to minimize the chance of losing the colony due to equipment malfunction. Periodically, clean up the growth chamber to remove dead insects and debris. Further, wipe the interior walls and shelving clean with a wet cloth followed by 70% ethanol to get rid of aphid honeydew that might have collected in the chamber.
  3. Under our experimental conditions, we have found that in order to obtain 20 successful replicates for data analysis of aphid feeding behavior, for each treatment at least twice that number of aphids have to wired and connected to the Giga 8d and a plant. 
  4. Wiring the insect is not difficult, but requires patience and practice. In order to connect the adult aphid to the insect electrode, any variation of the procedure can be used. For instance, Video 2 shows an alternate method of connecting an aphid to the insect electrode. The important point is to ensure that the glue and the gold wire do not hinder aphid movement. 
  5. Although it can be done manually, the accuracy and efficiency of calculating the EPG events can be greatly enhanced by using the various available programs Backus 1.0 (Backus et al., 2007), Sarria Workbook version 4.4.3 (Sarria et al., 2009), EPG-Calc 6.1 (Giordanengo, 2014), Excel macro JKL 2.0 (www.epgsystems.eu) and Ebert 1.0 (Ebert et al., 2015).
  6. Although the EPG protocol described here is for uninfested aphids, it can also be utilized to study the effect of viral infections on GPA feeding behavior.

Acknowledgments

We would like to thank Travis Isaacs and Sarah Moh for Video 1 and Video 2. Work in the Nalam laboratory was supported by faculty startup funds provided by Indiana University-Purdue University Fort Wayne. Work in the Louis laboratory was partially supported by the Nebraska Agricultural Experiment Station with funding from the Hatch Act (Accession # 1007272) through the USDA National Institute of Food and Agriculture. Work in the Shah laboratory was supported at varied times by grants from the National Science Foundation and the US Department of Agriculture. All the authors declare no conflict of interest.

References

  1. Backus, E. A., Cline, A. R., Ellerseick, M. R. and Serrano, M. S. (2007). Lygus hesperus, (hemiptera: Miridae) feeding on cotton: new methods and parameters for analysis of nonsequential electrical penetration graph data. Ann Entomol Soc Am 100(2): 296-310.
  2. Blackman, R. L., and Eastop, V. F. (2000). Aphids on the world’s crops: an identification and information guide. Chichester: John Wiley and Sons.
  3. Ebert, T. A., Backus, E. A., Cid, M., Fereres, A. and Rogers, M. E. (2015). A new SAS program for behavioral analysis of electrical penetration graph data. Comput Electron Agr 116(C): 80-87.
  4. Giordanengo, P. (2014). Epg-Calc: a PHP-based script to calculate electrical penetration graph (EPG) parameters. Arthropod-Plant Inte 8(2): 163-169.
  5. Kennedy, J. S., Day, M. F., and Eastop, V. F. (1963). A conspectus of aphids as vectors of plant viruses. London: Commonwealth Institute of Entomology pp: 114.
  6. Louis, J. and Shah, J. (2013). Arabidopsis thaliana-Myzus persicae interaction: shaping the understanding of plant defense against phloem-feeding aphids. Front in Plant Sci 4: 213.
  7. Louis, J., Singh, V. and Shah, J. (2012). Arabidopsis thaliana-aphid interaction. Arabidopsis Book 10: e0159.
  8. Matthews, R. E. F. (1991). Relationships between plant viruses and invertebrates. Plant Virology (3rd edition). In: Matthews, R. E. F. (Ed.) San Diego, CA: Academic Press pp: 520-561.
  9. Nalam, V., Louis, J. and Shah, J. (2018). Plant defense against aphids, the pest extraordinaire. Plant Sci
  10. Salvador-Recatalà, V. and Tjallingii, W. F. (2015). A new application of the electrical penetration graph (EPG) for acquiring and measuring electrical signals in phloem sieve elements. J Vis Exp (101): e52826.
  11. Sarria, E., Cid, M., Garzo, E. and Fereres, A. (2009). Excel workbook for automatic parameter calculation of EPG data. Comput Electron Agr 67(1-2): 35-42.
  12. Tjallingii, W. F. (1985). Electrical nature of recorded signals during stylet penetration by aphids. Entomol Exp Appl 38(2): 177-186.
  13. Tjallingii, W. F. (2006). Salivary secretions by aphids interacting with proteins of phloem wound responses. J Exp Bot 57(4): 739-745.

简介

蚜虫构成了一大群半翅目,它们使用细长的探针进入植物的筛子元素,从而消耗大量的韧皮部汁液,从而剥夺了植物的光合同化物。 一些蚜虫也传播植物的病毒性疾病。 桃蚜(Myzus persicae)Sülzer,俗称绿桃蚜(GPA),是一种多食性昆虫,寄主范围覆盖50个植物科,被认为是植物的三大害虫之一。 拟南芥与GPA之间的相互作用被用作研究植物 - 蚜虫相互作用的模型病理系统。 在这里,我们描述了我们的实验室用于饲养GPA的方案,以及用于研究拟南芥的GPA性能的无选择和繁殖生物测定。 此外,我们描述了电穿透图(EPG)技术的程序,以监测GPA在拟南芥上的摄食行为。

【背景】蚜虫是植物的重要害虫,它们利用它们的口器被改造成管心针,以从筛子元件中去除韧皮部汁液。作为其饲养过程的一部分,蚜虫将唾液沉积到植物组织中。虽然一些唾液成分引发植物防御,但其他驯鹿成分则操纵宿主生理学以使昆虫受益,包括抑制植物防御(Nalam et al。,2018)。植物利用各种防御来控制蚜虫侵扰。这些包括抗生素,它对蚜虫的生长,发育和繁殖以及影响昆虫行为(包括摄食行为)的抗病毒产生不利影响。当探针尖端在植物细胞内时,以及当探针尖端在筛子元件中时,这些防御在各个步骤施加,包括在细胞表面,在昆虫探针的叶组织细胞间穿透期间(Nalam 等人,,2018)。绿桃蚜(GPA), Myzus persicae Sülzer,是许多科的植物的重要害虫,包括十字花科,茄科,葫芦科,蔷薇科,菊科,锦葵科,苋科(Blackman和Eastop,2000) )。此外,GPA还传播了几种病毒性疾病(Kennedy et al。,1963; Matthews,1991)。在过去的十年中,属于十字花科的拟南芥与GPA之间的相互作用已被越来越多地用于研究植物 - 蚜虫的相互作用(Louis et al。 ,2012; Louis和Shah,2013)。这种病理系统促进了对决定植物 - 蚜虫相互作用结果的生理和分子过程的理解,包括植物防御机制及其对昆虫种群增长,繁殖力和行为的影响(Louis et al。 2012;路易斯和沙阿,2013年)。直流(DC) - 电穿透图(EPG)系统测量电动势(EMF)信号和蚜虫探针穿透引起的电阻波动,为监测植物的蚜虫摄食行为提供了一种灵敏的方法(Tjallingii,1985) ; Salvador-Recatalà和Tjallingii,2015)。当蚜虫探针插入细胞间时,电压为正,当插入细胞内时,电压为负(Tjallingii,2006)。不同的EPG波形图案表示昆虫参与的不同活动。此外,每种波形的持续时间提供了植物基因型和/或处理对昆虫摄食行为的影响的定量测量,包括昆虫从筛元件摄取所花费的时间。然而,尽管拟南芥 -GPA病原体系成功,但用于研究蚜虫种群增长,繁殖力和摄食行为的方案尚未详细描述。在这里,我们详细介绍了饲养GPA的十字花科植物群体的协议,监测GPA种群增长的无选择分析,监测昆虫繁殖率的繁殖力分析,以及监测拟南芥GPA摄食行为的EPG分析< / em>的。

关键字:绿桃蚜, EPG, 蚜虫的摄食行为, 植物 - 蚜虫互作, 筛管


第一部分:饲养绿桃蚜虫

绿蚜( Myzus persicae )菌落维持在萝卜( Raphanus sativus '早期猩红地球')和芥菜( Brassica juncea )的混合物上>'Florida Broadleaf')植物,其中拟南芥属于十字花科(Brassicaceae)。在十字花科(Brassicaceae)上,GPA通过释放活的无翅(若虫)若虫来无性繁殖。

材料和试剂

  1. 至。塑料标准平面1020托盘带底孔(Hummert International,型号:STE-1020-OPEN - 带孔,目录号:11-3000-1)
  2. 至。 Plastics Standard Flats 1020托盘无孔(Hummert International,型号:STE-1020-NH - NO HOLES,目录号:11-3050-1)
  3. 方形注塑塑料罐(4.5“[11.43厘米]宽x 3.75英寸[9.53厘米]高)底部有孔(国际温室,目录号:CN-SQK)
  4. 扭曲的关系
  5. Biohazard高压灭菌袋(Fisher Scientific,目录号:01-830D)
  6. Nalgene TM 聚丙烯重型灭菌盘(赛默飞世尔科技,目录号:6900-0020)
  7. 土壤混合物(Sunshine ®混合物#8,Sun Gro园艺,型号:Fafard ® -2)
  8. 萝卜种子(Radish Early Scarlet Globe)(Main Street Seed&amp; Supply,目录号:13307-13)
  9. 芥菜籽(Florida Mustard Broad Leaf)(主街种子和供应,目录号:12501-13)
  10. 绿桃蚜虫栖息地(标本号194保存在堪萨斯州立大学昆虫学和草原节肢动物研究博物馆)

设备

  1. 植物生长室(Percival Scientific,型号:AR-66L2)
    注意:编程为14/10小时(80-100μEm -2 sec -1 )/夜间光周期在22°C。
  2. 高压灭菌器

程序

  1. 将土壤放入高压灭菌袋中。打破任何肿块,加入自来水。揉捏土壤 - 水混合物,直至土壤均匀湿润。用塑料领带关闭袋子。用剪刀在塑料袋中制作四个小切口,以便在步骤2结束后从高压灭菌器中取出袋子后进行通风。&nbsp;
  2. 将含有土壤的袋子放入可高压灭菌的塑料托盘中,并在液体循环中在121℃/ 15psi下高压灭菌1小时。高压灭菌完成后,将带有污垢的托盘从高压灭菌器中取出,让土壤冷却至室温。最好让土壤在一夜之间冷却下来。
  3. 要准备种植萝卜和芥末的花盆,取一个带孔的扁平塑料托盘,放在没有孔的扁平塑料托盘内。将8个4.5英寸(11.43厘米)的花盆放入带孔的托盘中。用湿的高压灭菌土壤松散地填充这些花盆,确保将花盆填满顶部。&nbsp;
  4. 在每个盆的土壤顶部撒上30-40粒萝卜和芥末(参见图1)。


    图1. 4.5“(11.43厘米)锅中的萝卜和芥菜种子

  1. 用水慷慨地喷洒种子。
    注意:所有的种子都应该用水喷洒。如果种子保持干燥,它们就不会发芽。
  2. 将含有新鲜萝卜和芥菜种子的盆放在含有GPA定殖的萝卜和芥菜植物混合物的生长室中(图2)。将具有新鲜种子的托盘放置在含有GPA定殖的芥菜和萝卜植物的托盘附近。生长室应优选位于容纳区域,在该区域中不栽培其他用途的植物。


    图2.带有GPA的萝卜和芥菜植物(用黑色箭头表示)

  3. 萝卜和芥菜籽混合物应在2-3天内开始发芽。蚜虫将在几天内开始移植到新鲜植物中。
  4. 每周重复此过程以维持健康的GPA群体。&nbsp;
  5. 将不需要的植物和土壤倒入高压灭菌袋中,在液体循环中在121℃/ 15psi下高压灭菌1小时,然后处理。
    防止昆虫逃离封闭设施的步骤: 如果可行,将昆虫群体放在与通常种植植物的房间分开的房间内。为了限制昆虫从昆虫室传播出去,避免从昆虫室进入种植植物的房间。如果可行的话,避免穿着长袖的衬衫/实验服,或卷起袖子,这样昆虫就不会意外地爬到你的衬衫上,逃出了虫室。此外,在进行任何其他工作之前,请先洗手。

第二部分:无选择生物测定

无选择生物测定提供了一种简单的测定,用于比较不同基因型植物上的蚜虫种群生长。它提供了不同基因型植物抗性水平之间差异的量度。它还可用于比较各种处理(例如,化学品)对蚜虫种群增长的影响。无选择测定法测量抗菌和抗疟疾的综合效果。抗生素是宿主防御对昆虫生长,发育和繁殖的影响,而抗病毒反映了植物机制对昆虫行为的影响。稍后描述的电渗透图技术测量宿主基因型或处理对昆虫摄食行为的影响。

材料和试剂

  1. 至。塑料标准平面1020托盘带底孔(Hummert International,型号:STE-1020-OPEN - 带孔,目录号:11-3000-1)
  2. 至。 Plastics Standard Flats 1020托盘无孔(Hummert International,型号:STE-1020-NH - NO HOLES,目录号:11-3050-1)
  3. 饲养绿桃蚜的盆:方形注塑塑料盆(4.5“[11.43厘米]宽x 3.75英寸[9.53厘米]高度)底部有孔(国际温室,目录号:CN-SQK)
  4. 栽培拟南芥的盆:方形注塑塑料盆(3.5“[8.9 cm]宽x 3 7 / 8 ”[9.84 cm]高度)底部有孔(国际温室,目录号:CN-SQK)
  5. 土壤混合物(Sunshine ®混合物#8,Sun Gro园艺,型号:Fafard ® -2)
  6. 透明的乙烯基传播圆顶,适合1020平面(Hummert International,目录号:11-3360-1)
  7. 扭曲的关系
  8. Biohazard高压灭菌袋(Fisher Scientific,目录号:01-830D)
  9. Nalgene TM 聚丙烯重型灭菌盘(赛默飞世尔科技,目录号:6900-0020)
  10. 骆驼毛刷(2号或更小)(Fisher Scientific,General Data,目录号:15-183-35)
  11. 牙签
  12. 标签胶带0.5“(1.27 cm)宽(SP Scienceware - Bel-Art Products - H-B Instrument,目录号:F13481-0050)
  13. 细尖永久性酒精/防水实验室标记(VWR,目录号:52877-310)
  14. 绿桃蚜虫群(标本#194保存在堪萨斯州立大学昆虫学和草原节肢动物研究博物馆)
  15. Arabidopsis thaliana 种子加入哥伦比亚(但其他种质也应该没问题)
  16. 萝卜种子(Radish Early Scarlet Globe)(Main Street Seed&amp; Supply,目录号:13307-13)
  17. 芥菜籽(佛罗里达芥菜阔叶)(Main Street Seed&amp; Supply,目录号:12501-13)
  18. Peters 20:20:20通用肥料(Hummert International,目录号:07-5400-1)

设备

  1. 两个植物生长室(Percival Scientific,型号:AR-66L2)
    注意:生长室在22°C下编程为14/10小时(80-100μEm -2 sec -1 )/夜间光周期。一个是培育拟南芥所必需的,第二个室是蚜虫无选择测定所必需的。
  2. 高压灭菌器
  3. 冷藏室或冰箱(4-10°C)

程序

  1. 宣传拟南芥进行GPA实验
  1. 如上文第I部分程序中所述,对土壤进行高压灭菌并制备具有土壤的3.5“(8.9cm)盆。每个拟南芥基因型需要至少5个盆。在每个锅上粘上2-3厘米长的标签胶带,并用细尖防水标记记下植物基因型。
  2. 将充满土壤的盆放入带孔的托盘中。每个托盘最多可以容纳18个,3.5英寸(8.9厘米)的花盆。每个拟南芥基因型至少需要五个盆。
  3. 将0.1克彼得的20:20:20肥料溶解在1升自来水中。
  4. 用肥料溶液填充没有孔的托盘,深度为1-1.25英寸(2.54-3.81厘米)。
  5. 将装有土壤填充罐的托盘(带孔)放入装有肥料溶液的托盘中。肥料溶液将通过毛细作用上升通过土壤。当土壤顶部看起来潮湿(大约10-15分钟)时,抬起装有盆的顶部托盘并将其放置在一个小角度,使其位于下托盘的顶部边缘。让多余的肥料溶液排出到下托盘中。
  6. 土壤现在可以播种了。每种基因型需要至少10株拟南芥植物(每盆2只)。将相同基因型的两个拟南芥种子对角地放置在距离盆边缘1-1.25cm的对角线上。通过将牙签的湿润的尖端接触种子,可以一次一个地拾取种子。种子会粘在牙签尖上。现在可以通过将附着在牙签尖上的种子接触到湿润的土壤表面来将种子放置在土壤上。
    注意:不要将种子推入土壤中,每个盆中只有相同基因型的种子。
  7. 用透明的乙烯基传播圆顶盖住托盘。
  8. 将托盘放入冷藏室或冰箱中48小时(在此期间不需要照明)。这将使种子吸收水分并增加种子均匀发芽的机会。
  9. 在冷处理结束时,将覆盖有透明乙烯基传播圆顶的托盘和罐移动到生长室中。种子应该发芽,一对绿色的子叶应该在2-3天内出现。
  10. 一旦出现第一对真叶(通常在将盆放入生长室后8-9天),取下透明塑料圆顶。将植物留在生长室中。
  11. 每隔两周给植物施肥,将装有孔的托盘放入装有肥料溶液的托盘中(每升水加0.1克彼得20:20:20肥料)。

  1. 无选择生物测定设置和分析
  1. 当植物长达24-26天时(见图3),将它们带到昆虫室。


    图3.用于无选择生物测定的拟南芥植物

  2. 使用驼毛画笔轻轻地从萝卜/芥菜植物中轻轻捡起成虫无翅蚜虫(~1毫米大小),一次一个,并将它们放在每个拟南芥的中心植物。重复该过程,直至每株拟南芥植物上放置20只蚜虫。&nbsp;
  3. 将受侵染的植物放入生长室中。将花盆放在一起以确保相邻花盆中的花盆和植物不会相互接触。
  4. 8个小时后,计算每株植物的蚜虫(若虫和成虫)的总数。大多数蚜虫将位于叶片的背面(参见图4)。


    图4. 拟南芥背面的GPA

  5. 当比较两种基因型的昆虫种群大小时(参见图5),可以使用学生的 t - 测试来确定两种基因型上昆虫种群大小的平均值是否显着不同( P < / em>&lt; 0.05)。


    图5.代表性的无选择实验数据。没有选择实验用野生型和拟南芥的突变基因型对GPA表现出降低的抗性。在感染后2天采集蚜虫计数(成虫+若虫)。显示了每株植物上蚜虫的平均蚜虫数(n = 10)。误差线表示标准误差。星号表示两种基因型之间的值显着不同( P <0.05; t -test)。

  6. 在实验结束时,将植物和土壤倒入高压灭菌袋中,并在液体循环中在121℃/ 15psi下高压灭菌1小时,然后处理。

第三部分:繁殖力测定

繁殖力测定法用于确定GPA对个体基因型的繁殖能力,或用于比较不同基因型植物之间或用对照植物与化学品处理的植物之间GPA的繁殖能力。

材料和试剂

  1. 用于繁殖GPA和培育拟南芥和萝卜/芥菜植物,分别如上文第一部分和第二部分所述
  2. 骆驼毛刷(2号或以下)(Fisher Scientific,General Data,目录号:15-183-35)
  3. 14日龄拟南芥植物;每盆两个
  4. 培养皿100毫米宽x 15毫米深(Fisher Scientific,目录号:FB0875713)
  5. 萝卜种子(Radish Early Scarlet Globe)(Main Street Seed&amp; Supply,目录号:13307-13)
  6. 芥菜籽(佛罗里达芥菜阔叶)(主街种子和供应,目录号:12501-13)

设备

  1. 两个植物生长室(Percival Scientific,型号:AR-66L2)
    注意:生长室在22°C下编程为14/10小时(80-100μEm -2 sec -1 )/夜间光周期。一个是培育拟南芥所必需的,第二个室是蚜虫繁殖力测定所必需的。
  2. 高压灭菌器
  3. 冷藏室或冰箱(4-10°C)

程序

  1. 如上文第I部分和第II部分材料和试剂中所述,准备土壤和植物拟南芥和萝卜/芥菜种子。&nbsp;
  2. 在开始使用拟南芥进行实验前一天,用驼毛画笔在健康的萝卜/芥菜植物上放置几个成虫(~1-1.5 mm)昆虫。&nbsp;
  3. 第二天,用驼毛画笔将新出现的若虫收集在培养皿中。
  4. 使用油漆刷在每个拟南芥植物上释放两只1日龄的若虫。每个若虫都在另一片叶子上释放。每种基因型需要至少10株拟南芥植物。
  5. 将受侵染的植物放入生长室中。大多数这些若虫的大小会增加,并在大约6-8天内开始繁殖。繁殖力测定将确定这些(母)蚜虫产生的若虫的数量。
  6. 从第4天开始,每2天检查一下每株植物是否有新出现的若虫。计算若虫的数量并丢弃它们,只留下植物上的母蚜虫。
  7. 在拟南芥植物上释放母蚜后,继续计算新出现的若虫数量,持续17-18天。
  8. 确定在实验期间从每株植物中回收的若虫的总数。以每只蚜虫每天释放的若虫的平均数量表示的繁殖力用下式计算:

    繁殖力= N÷2(D)

    其中,N是在实验期间从每株植物中回收的新出现的若虫的总数,“D”是实验中的总天数。数字2表示在实验开始时在每个拟南芥植物上释放的昆虫数量。
  9. 当比较两种基因型的GPA繁殖力时(图6),可以使用学生的 t - 测试来确定两种基因型上的繁殖力值是否显着不同( P < 0.05)彼此。


    图6.蚜虫繁殖力测定。 显示了拟南芥野生型和突变基因型(n = 10)在18天内每天每只母蚜释放的若虫的平均数量。误差线表示标准误差。星号表示两种基因型之间的值明显不同( P <0.05; t -test)。

  10. 在实验结束时,将植物和土壤倒入高压灭菌袋中,并在液体循环中在121℃/ 15psi下高压灭菌1小时,然后处理。

第四部分:研究绿桃蚜虫摄食行为的电穿透图

电穿透图(EPG)测量蚜虫在各种喂养活动上花费的时间。它可用于确定植物基因型和/或环境因素(例如,化学处理)对蚜虫摄食行为的影响。 EPG设置的卡通图如图7所示。在EPG中,一个非绝缘电极,即植物电极,放置在植物生长的土壤中,从而使植物以极低电压,低电流强度通电当前。昆虫电极一端含有极细且柔韧的金线,粘在蚜虫的背部(背部)。金线的另一端连接到铜线,铜线又连接到黄铜钉上,黄铜钉连接到放大器。当蚜虫在拴在昆虫电极的金线上的植物表面上行走时,该开关在该电路中打开。然而,当探针接触带电设备的导电组织时,发生潜在的下降并且产生反映不同活动的独特波形图案,包括(i)非探测阶段(基线),(ii)当插入管心针时的路径阶段叶子,但不在韧皮部,(iii)第一次探测的时间,(iv)当昆虫从筛子元件喂食时的筛元素阶段(SEP),(v)到第一个SEP的时间,和(vi)木质部阶段。下面我们描述昆虫探针的制备步骤,昆虫电极与蚜虫的附着,数据采集和分析。


图7. EPG设置示意图 EPG监视器连接一根细金线(昆虫电极),可以让蚜虫畅通无阻地移动。将作为植物电极一部分的输出线(硬铜线)插入盆的土壤中,植物在该土壤中扎根。植物电极的另一端连接到EPG监测器。该设备通过低电压,低电流电流充电。一旦蚜虫开始以植物为食,蚜虫探针与电气化植物接触,电路将关闭,电流将流过昆虫并进入监测器,从而产生不同的波形。 Nick Sloff的插图,经路易等人的许可(2012)拟南芥 Book e0159。 doi:10.1199 / tab.0159, www.arabidopsisbook.org )。

材料和试剂

  1. 用于繁殖GPA和培育拟南芥和萝卜/芥菜植物,分别如上文第I部分和第II部分所述。
  2. 细金线Ø18μm(厚度范围10-20μm;可在 https://www.epgsystems.eu获取/ EPG /产品)
  3. 黄铜连接器针脚/钉子,Ø1.2mm(配有Giga-8d EPG系统; http://www.epgsystems.eu/ )
  4. 铜线Ø0.2mm(AliExpress,型号:YT1303)&nbsp;
  5. 可调节的旋转夹具用于固定EPG探头(EPG系统,附带Giga-8d EPG系统; http://www.epgsystems。欧盟/ )
  6. 铅焊辊(可从当地五金店购买)
  7. 培养皿100毫米宽x 15毫米深(Fisher Scientific,目录号:FB0875713)
  8. 骆驼毛刷(2号或以下)(Fisher Scientific,General Data,目录号:15-183-35)
  9. 解剖针(Thermo Fisher Scientific,目录号:19010)
  10. 纸针或T针
  11. Kimwipes ®(KCWW,Kimberly-Clark,目录号:34120)
  12. Andwin Scientific Miracloth孔径22-25μm(Fisher Scientific,目录号:NC9147303)
    制造商:Andwin Scientific,目录号:475855。
  13. 聚苯乙烯泡沫塑料/聚苯乙烯盒
  14. 用于繁殖GPA的萝卜/芥菜植物
  15. 21-28天拟南芥植物;每壶一个
  16. 导电银漆 - 胶体银(Ted Pella,目录号:16031,16034)
    注意:或者,准备自己的银胶,如 https://www.epgsystems.eu/downloads-install-files-manuals/file/24-add-ons-and-hints

设备

  1. 镊子
  2. GIGA-8直流放大器( http://www.epgsystems.eu/ )
  3. EPG探测器( http://www.epgsystems.eu/ )
  4. 植物电极( http://www.epgsystems.eu/ )
  5. 接地电缆和测试电缆(参见GIGA手册; http://www.epgsystems.eu/ )
  6. 立体解剖显微镜&nbsp;
  7. 用于记录和存储EPG波形实时监控的计算机
  8. 法拉第笼防止外部噪音*
  9. 用于接线蚜虫的真空操作板*
  10. 烙铁(可从当地五金店购买)将铜线连接到黄铜钉上
  11. 用于焊接的通风柜

注:*法拉第笼的结构和真空抽吸装置已在W.F.详细说明。 Giga 8d手册中的Tjallingii( https: //www.epgsystems.eu/downloads-install-files-manuals/file/24-add-ons-and-hints )。

软件

  1. Stylet + ( http://www.epgsystems.eu/ )
  2. JKI Macro for Excel( https:// www。 epgsystems.eu/downloads-install-files-manuals/category/4-epg-data-processing )
  3. 用于自动参数计算的Excel工作簿(Sarria et al。,2009)&nbsp;
  4. Ebert 1.0用于参数计算( http://www.crec.ifas.ufl埃杜/扩展/ EPG / sas.shtml )
  5. Microsoft Excel ®
  6. 方差分析(ANOVA)(SAS Institute Inc,SAS v5.1)
  7. Minitab ® 18.1用于非参数分析(Kruskal-Wallis检验和Mann-Whitney U检验)

程序

提供以下方案用于研究拟南芥和绿桃蚜(GPA), Myzus persicae 。

  1. 昆虫电极的制备
    昆虫电极(图8)由黄铜连接器针脚(Ø1.2mm)组成,铜线(2-4 cm长;Ø0.2mm)通过将导线焊接到黄铜连接器针脚的头部而连接(视频) 1; A部分)。使用银胶或胶体银涂料(视频1; B部分)将金线(2-4厘米长;直径18微米)连接到铜线的另一端。


    图8.昆虫电极的各种成分

    视频1
    1. 使用焊接螺栓,将一些焊接线熔化在坚硬,耐热的表面上。
    2. 将焊接液涂抹在黄铜接头销的头部,然后将黄铜接头销浸入熔化的焊接液中。
    3. 立即将带有焊接液的黄铜连接器针头与2-4厘米预切割铜线的一端接触(视频1; A部分)。
    4. 将铜线固定到位,直到焊接液冷却并固化在黄铜连接器针头上,与铜线形成牢固的粘合。
      注意:上述步骤A1-A4应在通风橱下进行。
    5. 将铜线的自由端浸入含有银胶或胶体银涂料的管中,以便在自由端形成精细的胶水/胶体涂料护套。
      注意:为了确保银颗粒和胶水均匀分布,请在使用前彻底摇匀银胶/胶体涂料。
    6. 将铜线的胶水/涂料端接触2-4厘米预切割金线的末端,然后轻轻提起金线。或者,在铜线上捻几圈金线并在其上涂上银色胶/涂料(视频1; B部分)。
    7. 使用解剖针,轻轻操纵金线,使胶水/涂料分布,使铜线与金线一端约0.5厘米重叠(视频1; B部分)。
    8. 在使用解剖针的尖端施加第二层胶/涂料之前,使胶/涂料完全干燥,以在铜线和金线之间形成牢固的粘合。此过程也是为了确保铜线和金线之间没有胶水/油漆接触部分。
    9. 胶水/涂料干燥后,昆虫电极就绪,可以存放在发泡胶/聚苯乙烯盒中。
      注意:昆虫电极可以使用立体解剖显微镜或在实验室工作台上充分照明来制备。

  2. 准备用于布线的蚜虫
    从蚜虫群体中收集足够的成虫蚜虫(至少10-15只用于制备8只昆虫电极接线的昆虫)并将它们转移到培养皿(100毫米宽×15毫米深)。
    在接线之前,蚜虫被挨饿一小时以确保在EPG实验开始时进食。
    注意:Giga-8系统可以同时监测最多8只蚜虫的摄食行为。因此,要获得15-20只蚜虫的读数,实验必须在多天内进行。

  3. 将蚜虫连接到昆虫电极
    上述昆虫电极(图8)用银胶/胶体涂料与蚜虫连接(图9A和9B)。对于以下程序,使用立体解剖显微镜将极大地简化该过程。可以利用真空抽吸装置将蚜虫保持在适当位置,同时将昆虫电极连接到蚜虫的背部。或者,可以将蚜虫放在KimWipe ®或MiraCloth ®上,以降低蚜虫的流动性。


    图9.有线绿桃蚜虫。 A.卡通描绘昆虫电极的金线部分,附着在GPA的背部,带有银色涂料/胶水。 B.通过金线和银色涂料/胶水连接到昆虫电极的GPA图像。

    1. 通过彻底摇动管子,确保可以获得银胶/胶体涂料的光滑乳液。
    2. 用骆驼毛油漆刷从培养皿中提起蚜虫,这些蚜虫成虫蚜虫正在饥饿。
    3. 使用针/解剖针,在成年蚜虫的背部涂上一小滴胶/胶体银涂料(图9A和9B)。让胶水/胶体银漆干燥几分钟。确保胶水/胶体银不会涂在昆虫的头部,触角或腿上。
    4. 一旦第一滴胶水/胶体条状涂料干燥后,在蚜虫背面的同一位置涂上第二滴胶水/胶状棉条涂料。
    5. 立即将昆虫电极金线的自由端插入第二滴胶/胶体银漆(视频2)。确保金线的另一端指向昆虫的上方并且不妨碍蚜虫的腿或触角的移动。

      视频2
    6. 将昆虫电极固定到位,直到胶水/胶体涂料干燥。这个过程需要几分钟,在此期间建议蚜虫和昆虫电极的移动最小。
      注意:如果胶水/胶体银漆涂在天线或蚜虫腿上或昆虫电极的金线没有附着,丢弃蚜虫并用新鲜的蚜虫重复该过程。避免在蚜虫背面添加第三滴胶水/胶体涂料。
    7. 此时,关闭抽吸装置(如果使用一个)或将蚜虫抬离立体解剖显微镜的载物台。驼毛涂料刷可用于帮助提升蚜虫。
    8. 然后将带有附着蚜虫的昆虫电极储存起来直到使用,方法是将黄铜连接器针头端插入聚苯乙烯泡沫塑料/聚苯乙烯块中(视频3)。

      视频3
    9. 重复步骤C1-C8,直到准备好EPG实验所需的所有蚜虫并准备就绪。

  4. 工厂访问
    1. EPG的植物应在实验前一天充分浇水。将拟南芥植物置于法拉第笼中的非导电表面上(图10A和10B)。非导电表面的示例包括培养皿,来自运输容器的纸板或切成适当大小的泡沫塑料块以放置在盆下。
    2. 如果使用Giga-8的所有8个通道,则在法拉第笼中排列8个拟南芥植物,使得叶子或花盆的任何部分都不会相互接触。
    3. 将植物电极插入每个盆的土壤中,尽可能靠近盆底,对植物根部造成的损害最小。
      注意:植物电极( http://www.epgsystems.eu/ )由铜组成电线(10-12厘米长;Ø0.6毫米)连接到电缆和连接器引脚,用于连接到Giga-8系统。
    4. 将带有蚜虫的昆虫电极的黄铜针插入EPG探头的输入连接器。昆虫的正确位置应使腿应位于植物表面的行走位置。
    5. 提起EPG探头(图10C),使昆虫不与植物表面接触,然后将剩余的准备好的昆虫电极与有线蚜虫连接到其余通道。
    6. 如果需要,使用旋转夹来调节EPG探头的高度(图10C)。
    7. 一旦连接了所有昆虫电极并且蚜虫悬挂在所选叶子/植物上方几厘米处,使用Stylet + 软件开始数据采集(使用该软件的详细方法可以在在 https上的Stylet + 手册: //www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software )。
    8. 将昆虫一次降低到植物表面并确保蚜虫能够将其腿放在植物表面上并且有足够的松弛度以使蚜虫四处移动以找到适合探针穿透的位置(图10D)。


      图10. EPG设置。 A.包含Giga-8设置的法拉第笼; B.关闭Giga-8装置,监测拟南芥上五种GPA的活性;光盘。由蚜虫电极附着到Giga-8系统的GPA的特写视图。
      注意:有些用户可能想要增加昆虫电极中金线的长度,以便为蚜虫移动留出足够的空间。然而,较长的金线会导致植物结构上的缠结,从而导致EPG记录期间的噪音。

    9. 一旦蚜虫开始探测,可以调整放大器设置(详细信息可以在Giga 8d手册中找到: https://www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software )。

  5. 重要的考虑因素
    要获得样本间差异最小的数据,应考虑以下因素。
    1. 最佳EPG记录时间:对于拟南芥的GPA喂养,最佳记录时间为8小时。可以延长录制时间。需要进行试验性实验才能准确确定EPG录音的持续时间。
    2. 植物年龄:确保植物的年龄尽可能接近。为了实现这一目标,遵循交错种植,使实验的植物具有统一的年龄。
    3. 蚜虫喂养的场所:如果可能并且您的EPG设置允许,将连接昆虫电极的蚜虫放在每株植物的同一叶子上即,避免将蚜虫放在不同的叶子上年龄。
    4. 时间:蚜虫表现出与任何其他生物体一样的昼夜节律,蚜虫的摄食行为可能随时间而变化。避免在夜间设置和运行EPG实验。努力在一天的同一时间进行所有实验。
    5. 复制次数:适当的重复次数应该能够补偿个体蚜虫之间的行为差异。 20个成功的重复是理想的,但至少15个是所有实验必不可少的。

  6. EPG波形分析
    Stylet + 分析(Ana)用于EPG波形分析。通过GPA对拟南芥植物进行饲喂,发现存在9种不同的波形:A,B,C(通路期),pd(潜在降低),E1和E2(韧皮部期或筛选元素期,SEP) ),G(木质部相),F(出轨探针相)和NP(非探测)(图11)。有关波形特征的更多详细信息,请访问: https://www.epgsystems.eu/downloads-install-files-manuals/category/4-epg-data-processing 。

    1. 波形的描述
      1. 在EPG实验期间,可以观察到当没有探针穿透(图11)并且蚜虫探针缩回并且不位于任何宿主组织中时的不活动期。这段时间称为非探测阶段(NP)。


        图11.在5小时内以拟南芥野生型登录哥伦比亚植物为食的GPA的代表性EPG波形模式。韧皮部或筛选元素相(SEP),木质部阶段(XP);确定了途径阶段(PP)和非探测阶段(NP)。

      2. 波形A,B和C(图12A)表示探针在表皮和叶肉组织中的位置。 A波形表示表皮中存在蚜虫管心针,B波形表示表皮或叶肉中存在管心针,C波形表示任何组织中管心针的存在。这三个波形重叠,很难分开。因此,它们作为“通路相位”或“通管路径”集中在一起,并在EPG分析中标记为波形C(图12A)。通路阶段(PP)或探针通路阶段,被称为探针穿透阶段,不是韧皮部或木质部阶段,包括各种探针穿透行为,包括细胞间探针前进和退缩,以及探针短暂的细胞内穿刺提示。正是在这个探测阶段,昆虫试图找到它的主要摄取部位(即,筛子元素),并做出关于宿主接受或拒绝的决定。
      3. 潜在的滴剂或pd代表管心针对植物细胞的刺穿(图12A)。 pd波形可以进一步细分为II-1,II-2和II-3(图12B)。如果实验涉及研究蚜虫接种持久性病毒,这些特征很重要。如果研究问题不需要,则不需要标记这些波形。


        图12.路径相的蚜虫波形,包括潜在的下降。 A.路径阶段由A,B和C波形组成一个类别,如CB A潜在下降(pd)显示II与3分阶段(II-1,II-2和II-3)。

      4. 当探针尖端处于韧皮部筛网元件中时发生SEP,韧皮部分是蚜虫吸收韧皮部汁液的部位。 SEP由E1(图13A和13B)和E2(图13A和13C)组成。在E1期间,蚜虫探针位于筛子元件中,并且蚜虫正在积极地垂涎,可能是为了抑制宿主反应。在E2期间,蚜虫探针位于筛子元素内,蚜虫正在摄取韧皮部汁液。&nbsp;


        图13.筛选元素相(SEP)的蚜虫波形。 A. SEP阶段显示E1和E2;公元前。分别扩大了E1和E2的视图。

      5. 在G波形期间(图14),蚜虫探针位于木质部,蚜虫正在摄取木质部汁液。


        图14.木质部饲喂/摄取阶段的蚜虫波形(G)

      6. 波形F指的是脱轨的探针(数据未显示),这意味着蚜虫探针遇到穿透困难。在此阶段,蚜虫管心针位于宿主组织中,但由于探针力学的困难,蚜虫不参与进食行为。

    2. 波形分析
      1. 可视化地执行各种波形的识别,并使用Stylet + 软件对波形进行标记(使用该软件的详细方法可在以下位置找到: https://www.epgsystems.eu/downloads-install-files-manuals/category/7-hard-software )。
      2. Stylet + 允许用户创建分析网格并将分析数据存储在下游数据处理所需的“.ana”文件中。
      3. 如果波形分析显示蚜虫花费了>在Np + F + G活动中70%的录音时间,丢弃蚜虫。

  7. EPG事件的计算
    EPG事件的长度和用于探索昆虫摄食行为的参数的复杂性使得EPG数据的分析成为耗时的过程。自动计算大量EPG参数的能力大大减少了所涉及的时间,提高了效率并提供了蚜虫昆虫探测和摄食行为的准确视图。几个已发布程序的可用性:Backus 1.0(Backus et al。,2007),Sarria Workbook version 4.4.3(Sarria et al。,2009),EPG-Calc 6.1(Giordanengo,2014),Excel宏JKL 2.0( www.epgsystems.eu )和Ebert 1.0(Ebert < em> et al。,2015)对于加快处理EPG实验期间产生的大量数据的速度和准确性是有价值的。研究人员选择用于数据分析的程序选择取决于实验设置,昆虫植物系统和软件工具的可访问性。 Ebert et al。(2015)对上面列出的三个程序(Backus 1.0,Sarria Workbook 4.4.3版和Ebert 1.0)进行了全面的回顾。

数据分析

从EPG记录获得的数据被认为是非参数数据,因为它们不符合单因素方差分析的假设,因此必须使用适当的统计检验进行分析,或者可以对数据进行转换以满足ANOVA的假设。两种分析方法是合适的:

  1. 无转换的数据分析:首先使用Kruskal-Wallis检验比较从G中列出的EPG分析工作表中获得的不同参数,这是一种无分布检验。可以使用Mann-Whitney U (MWU)测试进一步评估处理之间显示显着差异(α = 0.05)的参数(如果多于两个)。 MWU用于比较两种处理之间的差异,因此,必须在所有处理之间进行重复的MWU测试以确定显着差异(α= 0.05)。
    例如,如果您的EPG实验包含三种治疗方法:T1,T2&amp; T3和Kruskal-Wallis检验表明参数X的三种处理之间存在显着差异( P ≤0.05),然后确定哪些处理彼此显着不同,MWU测试是执行T1对T2,T1对T3和T2对T3。&nbsp;
  2. 使用秩变换的数据分析:为了使用ANOVA分析从EPG记录获得的不同参数,可以对数据进行等级变换。秩变换意味着每个参数的每个处理的活动值按排名顺序排列。为了进行这种转换,使用MS Excel中的RANK.AVG功能以顺序方式排列所有处理的每个参数的数据。此函数返回数字列表中数字的等级,如果多个值具有相同的等级,则返回平均等级。然后可以使用ANOVA(SAS或Minitab)分析每个参数的等级变换数据以确定处理之间的差异。显示 P 值≤0.05的参数可以进行事后检验,例如Tukey的“诚实显着性差异”检验,以确定不同治疗方法之间的差异。
    注意:Ebert 1.0是一个SAS程序,它提供EPG变量的数据编译和统计分析。使用混合模型方差分析进行数据分析(Ebert等,2015)。&nbsp;

笔记

  1. 拟南芥植物不应过度淹没。过度浇水增加了土壤中藻类和真菌生长的机会。最好只在土壤表面出现干燥的第一个迹象时进行分灌溉。这也将促进拟南芥的健康生长。
  2. 在单独的生长室中维持GPA群体的备用,以最大限度地减少因设备故障而失去菌落的机会。定期清理生长室,清除死亡的昆虫和碎片。进一步擦拭内墙并用湿布擦拭干净,然后用70%乙醇清除可能已收集在室内的蚜虫蜜露。
  3. 在我们的实验条件下,我们发现为了获得20个成功的重复数据进行蚜虫摄食行为的数据分析,每次治疗至少两次蚜虫必须连接并连接到Giga 8d和植物。&nbsp; < br />
  4. 连接昆虫并不困难,但需要耐心和练习。为了将成虫蚜虫连接到昆虫电极,可以使用任何程序的变化。例如,视频2显示了将蚜虫连接到昆虫电极的另一种方法。重要的是确保胶水和金线不会妨碍蚜虫的移动。&nbsp;
  5. 尽管可以手动完成,但通过使用各种可用程序Backus 1.0(Backus et al。,2007),Sarria Workbook 4.4.3版,可以大大提高计算EPG事件的准确性和效率。 (Sarria et al。,2009),EPG-Calc 6.1(Giordanengo,2014),Excel宏JKL 2.0( www.epgsystems.eu )和Ebert 1.0(Ebert et al。,2015)。
  6. 虽然这里描述的EPG协议适用于未感染的蚜虫,但它也可用于研究病毒感染对GPA摄食行为的影响。

致谢

我们要感谢Travis Isaacs和Sarah Moh的视频1和视频2.Nalam实验室的工作由印第安纳大学 - 普渡大学韦恩堡提供的教师启动资金支持。路易斯实验室的工作得到了内布拉斯加州农业实验站的部分支持,该实验站通过美国农业部国家粮食和农业研究所的Hatch法案(加入号#1007272)提供资金。 Shah实验室的工作在不同时期得到了美国国家科学基金会和美国农业部的资助。所有作者都声明没有利益冲突。

参考

  1. Backus,E.A.,Cline,A.R.,Ellerseick,M.R。和Serrano,M。S.(2007)。 Lygus hesperus ,(半翅目:Miridae)觅食棉花:用于分析非连续电渗透图数据的新方法和参数。 Ann Entomol Soc Am 100(2):296-310。
  2. Blackman,R.L。和Eastop,V。F.(2000)。 关于世界作物的蚜虫:识别和信息指南。奇切斯特:John Wiley和Sons。
  3. Ebert,T。A.,Backus,E。A.,Cid,M.,Fereres,A。和Rogers,M。E.(2015)。 用于电渗透图数据行为分析的新SAS程序。 Comput Electron Agr 116(C):80-87。
  4. Giordanengo,P。(2014)。 Epg-Calc:基于PHP的脚本,用于计算电穿透图(EPG)参数。 Arthropod-Plant Inte 8(2):163-169。
  5. Kennedy,J。S.,Day,M。F.和Eastop,V。F.(1963)。 A蚜虫作为植物病毒载体的概述。 伦敦:英联邦昆虫学研究所 pp:114。
  6. Louis,J。和Shah,J。(2013)。 Arabidopsis thaliana - Myzus persicae 互动:塑造对植物防御韧皮部蚜虫的理解。 植物科学前沿 4:213。
  7. Louis,J.,Singh,V。和Shah,J。(2012)。 Arabidopsis thaliana -aphid interaction。 拟南芥预订 10:e0159。
  8. Matthews,R。E. F.(1991)。植物病毒与无脊椎动物之间的关系。 植物病毒学(第3版)。在:Matthews,REF(Ed。) San Diego,CA:Academic Press pp:520-561。
  9. Nalam,V.,Louis,J。和Shah,J。(2018)。 植物防御蚜虫,非凡的害虫。 植物科学
  10. Salvador-Recatalà,V。和Tjallingii,W。F.(2015)。 电渗透图(EPG)的新应用,用于采集和测量韧皮部筛元素中的电信号。 J Vis Exp (101):e52826。
  11. Sarria,E.,Cid,M.,Garzo,E。和Fereres,A。(2009)。 用于EPG数据自动参数计算的Excel工作簿。 Comput Electron Agr < / em> 67(1-2):35-42。
  12. Tjallingii,W.F。(1985)。 蚜虫穿透探针时记录信号的电气特性。 Entomol Exp Appl 38(2):177-186。
  13. Tjallingii,W.F。(2006)。 蚜虫与韧皮部伤口反应蛋白质相互作用的唾液分泌物。 J Exp Bot 57(4):739-745。
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引用:Nalam, V., Louis, J., Patel, M. and Shah, J. (2018). Arabidopsis-Green Peach Aphid Interaction: Rearing the Insect, No-choice and Fecundity Assays, and Electrical Penetration Graph Technique to Study Insect Feeding Behavior. Bio-protocol 8(15): e2950. DOI: 10.21769/BioProtoc.2950.
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Danny Jones
ST John
Great info it was.
2019/1/21 0:48:13 回复
Jyoti Shah
Department of Biological Sciences and the BioDiscovery Institute, University of North Texas, USA, USA,

Thank you.

2019/1/22 20:30:07 回复