What is GC-EAD?
GC-EAD is coupled gas chromatography - electroantennographic detection. It is an analytical procedure that permits the rapid identification of compounds in complex mixtures that stimulate the olfactory sensilla of an insect. In other words, it can tell you what specific chemicals an insect can smell (and, to some degree, ones it can’t), and it can use odors derived directly from natural sources. This information can be used to discover potentially useful compounds—such as sex pheromones—that alter the behavior of insects.
In the 1950s, it was discovered that the voltage between the tip and the base of an insect’s antenna changed measurably when the antenna was exposed to odors of biological significance for the insect. This voltage is thought to represent the summed potentials of multiple responding olfactory neurons within the antenna, and the amplitude of the voltage roughly corresponds to an insect’s sensitivity to a particular compound. The voltage change does not reliably indicate whether a compound will influence the behavior of an insect or what the behavior might be, and it is quite common for compounds that produce strong antennal responses to have no observable behavioral effect. However, the presence of strong antennal responses (or responses to very low concentrations) indicates a greater probability that a compound will later be found to influence the behavior of an insect. Hence, antennal assays can assist in screening through the hundreds of odors found in the environment of an insect to permit the identification of those most likely to have behavioral activity. This can help in prioritizing compounds for behavioral tests and can greatly speed the identification of compounds that can potentially be used to modify insect behavior in beneficial ways.
Compounds can be exposed to the antenna after being purified or synthesized in a separate process; this procedure for manually exposing an antenna to compounds one at a time is known as an electroantennogram (EAG), and has received wide use in entomological studies since the late 1960s. The GC-EAD takes the EAG to a higher level of sophistication and utility by using the antenna of an insect as a detector for a capillary-column gas chromatograph.
The gas chromatograph (GC) is an apparatus used for separating and determining the identity and relative abundance of compounds present in complex mixtures of volatile and or semi-volatile compounds (Figure 1). Mixtures are flash-evaporated into a stream of inert gas moving through a long (typically 10-100 m), narrow (typically < 1mm) tube (called the “column”) lined with a semi-solid wax or polymer.
As compounds are carried along in the stream of flowing gas, some possess greater chemical affinity for the column’s lining than others and will travel more slowly. As a result, compounds will become separated as they travel within the column and will exit at different times (the “retention time” of the compound). Separation of complex mixtures by means of the differential partitioning of compounds between a “stationary” phase (in this case, the column lining) and a “mobile” phase (the inert gas) is a mechanism common to all forms of chromatography. In gas chromatography, the speed at which a compound travels is also dependent on the temperature of the column (compounds move faster at higher column temperatures). For this reason, the column is housed in an oven whose temperature can be precisely controlled. The effluent of the GC column is delivered to a detector which produces a DC voltage whose amplitude is proportional to the concentration of compound eluting from the column at that moment. The most common and most generally useful detector is the flame ionization detector (FID) which is sensitive to all organic molecules. The output of the detector is normally displayed on an X-Y graph with the Y axis representing detector voltage and the X-axis representing time. With the same column and GC operating conditions, a particular compound will always elute with the same retention time. Hence retention time is a diagnostic character that can be used to identify individual compounds in mixtures of unknowns.
In GC-EAD, the effluent from the end of the column is split in two, with one portion of the effluent delivered to an FID and the other passed into a stream of purified air that is blown across an insect’s antenna (Figure 2). Electrodes (normally saline-filled glass needles) attached to the tip of the antenna and to the antenna’s base (or, alternatively, to the head or body of the insect) conduct DC voltages from the antenna to a high-impedance input amplifier which, in turn, feeds the signal to a graphical readout that simultaneously plots antennal voltage and FID voltage outputs against time (Figure 3). Synchronous voltage changes by both the FID and the antenna indicate olfactory sensitivity by the insect to the compound eluting at that particular retention time. The FID output can be used to confirm the presence, identity, and quantity of compounds exposed to the antenna while the antennal (EAD) output indicates presence/absence of olfactory sensitivity to eluting compounds and provides a relative measure of the intensity of olfactory stimulation.
Ultra-Fine Dissecting Probes
The dissecting probes above were fabricated for manipulating an antenna once it is mounted on the indifferent electrode. These tools are extremely helpful for inserting the end of a long filamentous antenna into the tip of the signal electrode. They are made by inserting the base of a minuten insect pin into the end of the wooden shaft of a cotton-tip applicator. A hole is initiated in the shaft with a heavier pin and the minuten is secured in place with glue (hot-glue was used for the probes shown here). The minuten can be bent into a variety of shapes for addressing specific needs.
Tool for Scoring (and Breaking) Pipette Tips
For the signal electrode to make “noise-free” contact with the tip of an antenna, it helps if the tapered end of the pipette is cut as cleanly as possible with no uneven or jagged edges. Crushing or breaking the pipette with a forceps almost never leaves a clean, circular opening. To prevent the glass from shattering, first score (“scratch”) the glass with the tool shown (above right), and then break the pipette tip off cleanly by gently grasping it with a forceps just beyond the scratches and twisting carefully (see Antennal Preparation). The scoring tool was constructed from fragments of a ceramic wafer (used for scoring GC capillary columns) that had been smashed with a hammer. Fragments with especially sharply-angled edges are inserted into a slit cut into one end of the wooden shaft of a cotton-tipped applicator and secured with a small amount of glue. The tool provides the precision and control needed for scoring the pipette tip at a specific, desired location
Storing Pulled Electrode Pipettes
The pipette storage device shown above was made by sticking a piece of adhesive-backed weather stripping to the bottom of a 10-cm diam. plastic petri dish and cutting a series of width-wise slits into the foam rubber. The pipettes are held securely within the slits, preventing damage to the sharpened ends. We use a forceps or a "triceps" for maneuvering the pipettes in and out of the slits. We got the idea from Eva Pettersson who used a strip of dental wax instead of the weather stripping.
We have found that the preparation method detailed below works well for antennae of most insects we have examined, and it provides a high degree of reproducibility and a longer antennal life than other methods we have attempted so far. It has been used successfully with Coleoptera (6 species), Hymenoptera (7 species) and Lepidoptera (1 species). It is our standard method for making “first attempts” with a new insect species because of its reliability and simplicity.
Its major advantage is that the tip of the antenna does not need to be cut or punctured, reducing the number of steps required while simultaneously improving the usable life of the antenna. When surfactant is included in the saline (0.02% v/v Triton X-100 in Beadle-Ephrussi ringers solution) the signal/noise ratios obtained with this method are equal to those obtained using cut antennae. The basic procedure is outlined in the Syntech® manual (Electroantennography: A practical introduction 1998 version, p. 7) but we have elaborated on it.
The steps are illustrated here with a preparation of the clerid bark beetle predator, Thanasimus dubius:
- The insect's head is excised with a #11 scalpel blade on a pad of filter papers moistened with distilled water. The tip of one saline-filled electrode is broken at a point where its diameter is slightly less than the foramen (the opening between head and thorax) and then inserted into the foramen until the head is firmly secured.
- The pipette/head is attached to the indifferent electrode holder while a second saline-filled electrode is secured to the recording electrode holder positioned opposite.
The tip of the recording electrode is broken off at a point where its internal diameter is slightly less than the maximum diameter of the antenna's (or the antennal club's) distal-most segment. The pipette break should be as smooth and symmetrical as possible. Clipping or crushing the pipette tip with a forceps invariably leaves a jagged edge or produces an opening of inappropriate size. For this reason we first gently scratch (“score”) the glass at the point where we want to break it using a piece of ceramic wafer secured in a wooden dowel (bottom left; also see Nifty Tools). Once scratch marks are visible on the glass, we very gently grasp the pipette tip with a sharp forceps just beyond the scratches and twist slowly in the direction opposite the scratches (bottom center). In most cases, the pipette will break cleanly leaving a smooth, circular opening (bottom right).
- The cut pipette tip is maneuvered with a micromanipulator until the saline just makes contact with the tip of the antenna (below). If the antenna is oriented perpendicularly or away from the tip, a small wire probe (see Nifty Tools) can be used to guide the tip of the antenna into the saline meniscus. Avoid any contact between the saline and the sides of the antenna. Once contact with the saline is achieved, the surface tension of the saline will maintain the antennal tip in contact with the electrode while the recording electrode is repositioned. (For most antennae, it is essential that the saline contain some wetting agent—such as Triton X-100 as described above—for the uncut antennal tip to adhere to the saline.)
- The recording electrode is moved away from the head to straighten the antenna and then moved in a roughly circular motion to gradually work the tip of the antenna into the pipette tip (bottom right). When finished, the antennal tip should effectively “plug” the opening of the pipette with its circumference, leaving a minimal amount of saline from the pipette in contact with the air (below).
- Below are some additional examples of this technique applied to antennae of other insects.
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