1. Aphid Rearing

Note: The choice of plant and aphid species for EPG recordings depends on the research aim. For studies on Arabidopsis thaliana, the aphid Brevicoryne brassicae is appropriate.

1. Rear B. brassicae aphids in a greenhouse on Brassica oleracea. Keep the plants used for aphid rearing in cages, in order to avoid contaminating other plants. Keep aphid-rearing plants and experimental plants (in our case B. oleracea and A. thaliana) in separate rooms, in order to avoid contamination of experimental plants with aphids.
2. Transfer aphids to fresh plants about every 2 weeks, before causing significant plant damage, or reaching overpopulation. Transfer 10-20 adult aphids to a fresh rearing plant to initiate a new colony.
3. Monitor rearing plants regularly for contamination by unwanted aphid species, other insect herbivores, aphid parasitoids, and fungi that may affect the health of the aphid colony.
4. Collect adult, wingless aphids up to one week after their final molt for EPG recording.
5. After the experiments, return the experimental plants that were not used to the growth chamber, as they often have some offspring that has been produced during the recording, which could inadvertently contaminate other plants.

2. Insect Wiring for EPG Recording

1. To make insect electrodes, obtain brass connector pins (nails, Ø 1.2 mm), thin copper wire (Ø 0.2 mm), very thin gold wire (Ø ca. 20 µm), water-based silver glue, a simple small soldering bolt with soldering fluid and resin-cored soldering wire, stereomicroscope with 10X magnification, small scissors or scalpel, two fine forceps, and a Styrofoam sheet or box. Note: A vortex mixer might be useful. Note: The step-by-step protocol to make electrodes is indicated in Figure 2.
2. For aphid handling and glue application, obtain: a small and soft watercolor camelhair brush (size 2 or smaller) and insect pins such as those used for insect collections, although a fine sewing needle or toothpick may work as well. Step 4 shows how to start EPG recording.
3. Step 1.
   ​Note: Steps 1 and 2 below show how to prepare insect electrodes minus the aphid. Step 3 shows how to connect an aphid to the electrode. Vacuum fixation of the aphid is recommended during wiring, but not always required for slow moving species (e.g., B. brassicae).
   1. Switch on the soldering bolt and melt some soldering wire at its tip (Figure 2A). Moisten the head of the brass connector pin with some soldering fluid (Figure 2B) and dip it into the melted soldering metal (Figure 2C).
   2. Apply a sheath of melted solder metal on one end of a 1-2 cm long piece of the thin copper wire (Figure 2D). Then bring the pin and copper wire together against the hot bolt (Figure 2E) and move them together away to cool and solidify (Figure 2F).
4. Step 2.
   1. Thoroughly shake (or vortex) the vial with silver glue for several minutes until a smooth emulsion is shown. Cut (scissors or scalpel) a few pieces of the gold wire (of approximately 1.5 cm in length) on the object plate of the stereomicroscope (Figure 2G).
   2. Take a brass pin with soldered copper wire (made in section 2.3) and dip the free end of the copper wire into the small silver glue reservoir that will have gathered at inside the lid of the vial after opening it (Figure 2H). Note: Only a small droplet is needed.
   3. Move the glue-dipped end of the copper wire to the piece of gold wire, while lifting one end to avoid smearing the glue onto the stereomicroscope object plate. Try to overlap copper and gold wire for a few mm (Figure 2I), distributing the glue along the overlap of the two wires.
   4. Wait until the glue has been dried enough to keep the wires united. Check the glue contact after drying and add some fresh glue with a small pin or other piece of copper wire if some parts of the joined wires show glue-free parts.
   5. After the insect electrode is ready, store it, for example inserted into a piece of Styrofoam.
      Note: The length of the gold wire will determine the freedom of movement of the aphid: if it is too short (less than 5 mm), the aphid may feel constrained and will not behave normally; if it is too long (>2 cm), the aphid will move freely. Aphids tend to move to the adaxial side of leaves, if allowed to. If the gold wire touches the leaf, the signal will be short-circuited.
5. Step 3.
   1. The aphid may be kept in place by means of light suction, using a vacuum; in this case, install the suction device under the stereomicroscope. Place the suction opening in the center of the field.
   2. Thoroughly shake the vial with silver glue for several minutes (or vortex) until a smooth emulsion is formed. Collect an aphid with the small brush.
   3. Switch on the suction device and mount the aphid on the suction opening (Figure 2J), with the back of the abdomen turned to the experimenter. With the fine brush, remove any surface wax from the abdomen (abundant in cabbage aphids).
   4. Open the glue vial and wet a pin with a very small droplet of silver glue (Figure 2K). Apply the droplet of the silver glue onto the back of the aphid's abdomen (Figure 2L-M). Let this droplet completely dry during several minutes, vigorously shake the glue vial again and add a second droplet of silver glue on top of the first. Note: While the silver glue is an electrical conductor, it does not cause significant damage to the insect’s cuticle.
   5. After closing the glue vial, insert the free end of the gold wire into the wet droplet and keep the wire still while allowing the glue to dry completely (Figure 2N). Avoid smearing glue onto legs or antennae and discard an aphid if this has happened.
   6. Switch off the suction fixation device and carefully lift the insect (Figure 2O). If needed, use a fine brush to assist in the lifting of the aphid from the suction device.
      ​Note: Wiring B. brassicae does not require a vacuum, as they can be wired on a piece of a precision laboratory tissue, the rough surface of which provides the aphid with enough grip so that it will not be lifted after applying a drop of wet glue to its abdomen. After glue drying one can lift the aphid from the tissue with the help of a fine brush.
   7. Insert the brass pin with the wired insect into the Styrofoam and if needed, continue wiring all other insects to be used for the EPG recording session.
      Note: These protocols for wiring aphids work well for us. The user may find his/her own method for wiring aphids.
6. Step 4.
   1. Put plants in the Faraday cage (Figure 2P) on a non-conductive support: use Petri dishes or a plate of glass or plastic.
   2. Insert a plant electrode into the soil of each pot. Insert the brass pin of the wired insect into the input connector of the EPG pre-amplifier (Figure 2Q). Note: the soil electrode does not correspond to the ground electrode used in other electrophysiological techniques. It has the offset voltage needed to adjust and compensate for electrode polarization voltages.
   3. On the interface of the acquisition software Stylet+, with fixed sample frequency of 100 Hz, enter a filename, specify the recording time, and write text to specify details of the experiment (treatment, plant/insect species, etc.) in Comment lines 2 and 3.
   4. Lower the insects onto a suitable landing area of the plant and start the recording session by clicking on the Start button of the acquisition software (Stylet+) interface.
      Note 1: a maximum of 8 channels can be used simultaneously in an EPG set up. One EPG-electrode or several EPG-electrodes per plant can be used.
      Note 2: when the focus of the study is the aphid behavior, start the recording before plant access of the aphids to avoid missing the first plant penetration activities.
   5. For studying the electrophysiological responses of SEs to stimuli, wait at least 10 min after the aphid has entered into phloem phase, in order to ensure that the aphid is in a sustained phloem ingestion phase, and that the signal baseline is stable. Only then, start any plant stimulation experiment.

Figure 2. Making EPG-electrodes with aphids or other hemipteran insects forelectrical penetration graph (EPG) recordings. Please click here to view a larger version of this figure.

Panels A-I, steps required to prepare EPG-electrodes minus the aphid. First, melt a piece of soldering metal on the tip of a soldering bolt (A). Then, dip the head of the brass pin into a drop of soldering fluid (B), and contact it with the melted metal at the soldering bolt tip (C). Immediately after this step, contact the end of a copper wire to the tip of the soldering bolt, in order to glue it to the head of the brass pin (E-F). With a scalpel or a blade, cut a piece of the gold wire (G). Dip the free end of the copper wire (joined at the other end to the brass pin) on the silver glue (H), and quickly join the gold wire to it (I) before the silver dries up. The gold wire is an excellent conductor, and can be polarized. In reality, in the majority of the cases the polarization is too small to be detected, and if so, it can be compensated for with the offset voltage (V).

Panels J-O, steps needed to connect an aphid (or other hemipteran insect) to the electrode. First, carefully lift an aphid with a fine watercolor brush and place it on the opening of the vacuum suction device (J). Turn on the vacuum pump and cover the air valve hole with a piece of paper to apply suction. Dip the tip of the insect pin into the silver glue (K), and put a small glue droplet on top of the aphid's abdomen, under a stereomicroscope (L-M). Within the next ~20 sec, before the silver glue droplet on the aphid dries, insert the end of the gold wire of the insect electrode into the wet droplet of silver glue, and keep it in place for 1-3 min, until the silver glue has completely air-dried (N). At this point, disable suction by removing the piece of paper that covers the air valve hole of the suction device and carefully remove the aphid, from the suction device: lifting the aphid after wiring often requires some help by a fine brush (O).

Panel P shows an overview of the entire EPG set up inside the Faraday cage, and Panel Q shows an overview of the plant-aphid combination for EPG. See section 2 above for a more detailed explanation of this process.

Small letters are labels referring to the items that one needs to make EPG-electrodes: a: soldering bolt; b: melted soldering metal; c: soldering fluid; d: brass connector pin (nail); e: copper wire; f: Ø 18µm gold wire; g: suction device; h: aphid; i: water-based silver glue; j: Faraday cage; k: plant electrode; l: input connector (BNC) of the EPG pre-amplifier.