A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

The overall goal of the following experiment is to study the long distance electrical signals transmitted via the flow sieve elements from an area being eaten by a caterpillar to an intact area in a plant. This is achieved by assembling an electrical penetration graph or EPG circuit that integrates a hemin insect, such as an aphid and a plant. The aphid inserts its style it into the plant, thus closing the circuit, and the EPG recording begins.

Eventually the aphid inserts its style it into a sieve element and salivates into it for a few minutes, followed by the long lasting ingestion phase, both the salivation or E one phase and the ingestion or E two phase contains small and rhythmic streaming potentials. Next, a caterpillar is placed on a leaf and left to feed from a leaf. While the EPG recorded aphid continues to feed from the flow in a neighbor leaf, the damage inflicted by the caterpillar to the mid vein will start an electrical signal that will be transmitted via the phem to the other leaves, including the leaf from which the EPG wired aphid is feeding.

The size of the signal can be measured by comparison with a calibration pulse of known size, which was applied previously during the E two or ingestion phase. The results show that Caterpillar mediated damage of a leaf consistently induces a complex and significantly large electrical signal in the flow of a neighbor leaf with a first slow component and a second and superimposed to the first fast component. The electrical penetration graph technique has been introduced in the 1960s.

EPG monitoring has substantially contributed to our knowledge of plant penetration by aphids and other hem insects. The main economic interest is in studying mechanisms of host plant resistance, as well as mechanisms of transmission of plant viruses. So I came up with a concept of using EPG as a method for intracellular electrophysiology in plants by making an association between EPG recordings and intracellular recordings that are made with conventional glass electrodes that are filled with potassium chloride in both EPC recordings and conventional recordings made with glass electrodes.

There are two voltage levels, a relatively high voltage level that corresponds to the intracellular position of the tip of the glass electrode or the tip of the AEs style, and a relatively low voltage level, which corresponds to the intracellular position of the glass electrode or of the AEs eyelet. In the case of EPG. When rearing aphids for a PG recordings, the choice of plant and aphid species depends on the research Aim for studies on the rapid opana, the aphid is appropriate, raise them in a greenhouse on the cages surrounding the rearing.

Plants must be made aphid and parasitoid proof using fine mesh screening with openings at less than a millimeter, but cage tops should be of transparent material to allow photosynthesis. Every two weeks, the aphid population must be moved to fresh plants. 10 to 20 adults are all that are needed, the remaining aphids can be disposed of.

For insect EPG recordings, a specialized electrode must be constructed from brass connector pins, a one to two centimeter length of copper wire, such as filaments from an electrical cord, and a piece of very thin gold wire of about 1.5 centimeters in length. Also, use a water-based silver glue and a simple small soldering bolt with soldering fluid and resin cord soldering wire. To begin melt a piece of soldering metal wire on the tip of a soldering bolt.

Then dip the head of the brass nail into a drop of soldering fluid. Immediately contact the tip of the copper wire to the tip of the soldering bolt, thus melting it to the brass pin. Now thoroughly shake or vortex the silver glue so the emulsion is not settled.

Open the bottle and dip the free end of the copper wire into the glue on the lid under a stereoscope. Join the gold mesh with an overlap of at least one millimeter and let the silver glue dry. Try not to smear the glue onto the microscope plate after the glue is dried, check the contact between the silver glue and the gold wire where parts of the joint wires are not glued.

Add fresh glue with a small pin or copper wire until the insect is ready. The completed electrode can be stored safely on styrofoam. To fix the aphid, use a simple house built vacuum device.

The suction is controlled by a small piece of paper over an inlet hole. Switch on the vacuum device and move it on the plate of the stereo microscope to center its suction tip. Collect an aphid on a small size two watercolor brush or smaller, and mount the aphid on its ventral side towards the suction tip For easy access to the dorsum.

Using the brush, remove any surface wax from the abdomen, which is abundant in the cabbage aphid. Now thoroughly shake or vortex the silver glue and using a fine insect pin, apply a small droplet of silver glue onto the aphids dorsum. Let this dry for two to three minutes.

Shake the vial again and apply a glue droplet on top of the dried first droplet. Then attach the gold wire of the prepared insect electrode into the second droplet of silver glue. While it is still wet, keep the wire in position held still while the glue dries.

If any glue happens to get on the legs or in any of the aphid, start over with the new animal. The insect electrode can be recycled. Once the glue has dried.

Switch off the suction and free the aphid from the vacuum device. The wired insect is now ready to be used for EPG recordings. Some aphids may be wired without vacuum fixation.

In this case, place the aphid on a piece of laboratory tissue, which is rough enough for the aphid to stay gripped. During glue application, a gold wire can be inserted into the first droplet of silver glue. If this droplet is too small or if it dries too quickly, the gold wire will not attach properly.

In that case, simply place a small droplet of silver glue on top of the first one and insert the gold wire, wait until the glue dries. Then lift the aphid from the tissue paper with the help of a fine brush. Store the wired aphids by inserting the brass nail into the styrofoam until it is needed.

To begin, put the experimental plants into the Faraday cage on a non-conductive support like a Petri dish. Now, insert a plant electrode into the soil of each pot. The plant electrode is not a ground electrode that needs to be collected to the Faraday cage.

Then insert the brass pin of the electrode to the EPG. Pre adjust the position of the plant and the insect until they are in contact with the insect on the desired position on the plant. Using the Style plus interface software, which has a fixed sample frequency of 100 hertz, enter a file name, specify the recording time and write text to specify details of the experiment in the comment lines.

Then start the recording via the software by clicking on the start button on the top left corner. Begin the experiment by lowering the wired aphid onto the plant up to eight channels, meaning eight wired insects can be recorded simultaneously by one EPG setup either one per plant or more aphids on one plant. When the aphid inserts its style it into the plant, the circuit is completed.

The fluctuating voltages of the EPG signal will appear on the computer screen, make adjustments of the plant voltage and gain for optimal recording. In most cases, it will take an aphid at least one hour to enter into the flow phase. Sustained flow feeding is indicated by at least 10 minutes of waveform E two.

At this point, the electrode is ready to acquire electrical signals from the sieve element, which should only be induced when the E two signal is stable. It is important to deliver one or more calibration pulses, which will be used later to estimate the size of the electrical signals. Cutting a leaf with scissors consistently induces a long distance electrical signal that is acquired by the EPG recorded aphid.

In a more realistic experiment, a hungry caterpillar is used as the wounding agent. Caterpillar inflicted damage consistently induces slow and fast electrical signals that travel to the other leaves and can be acquired with EPG electrodes. The noise generated by introduction of the caterpillar will not affect the electrophysiological response of the plant, but it may mask it to minimize this noise.

The experimenter should be grounded to test whether a feeding caterpillar induces electrical signals in the leaf being eaten. A hungry caterpillar is placed on the leaf that also holds the EPG recorded aphid when two different leaves were sequentially cut with scissors at the peral lamina junction. An EPG electrode placed on the mid vein of an intact leaf that was a neighbor to both leaves being cut detected similar electrophysiological responses from the same sieve element to these wounds.

These electrophysiological consisted of a slow depolarization and fast depolarization superimposed to the slow one. In another experiment, two leaves were also cut, but in this case, one was a neighbor to the leaf containing the EPG electrode, whereas the other was not a neighbor to the leaf with the EPG electrode. In this case, the single sieve element responded differently to the wounds.

The wound to the non neighbor leaf made first induced only a slow electrical signal, whereas the wound to the neighbor leaf induced a complex signal with slow and fast components. These results suggest that SIV elements are able to detect the location of the damage here using an aphid connected to the EPG system, intracellular recordings were made from a marginal sieve element in a leaf of an arabidopsis plant. Cutting a neighbor leaf induced an electrical signal that was transmitted to the sieve element in which the aphid stylet was inserted.

Unlike sieve elements in major veins, the sieve elements in marginal veins responded to remote damage with only a single slow depolarization wave that may correspond to the slow depolarization wave in central sieve elements. Electrical penetration graph or repeat is a method which can help answer key questions in the fields of plant electrophysiology and plant physiology such as, for instance, which are on channels, underlie the long distance electrical signals which travel from leaf to leaf, and most importantly, what are the roles of these signals in adapting the plants to their environment? After watching this video, you should have an idea how to prepare plants and insects for EPG.

Recording insect wiring seems complicated, but we have shown that it is rather simple and it may encourage you to implement it in your own research.