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.  

                                  

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        Figure 1. A basic gas chromatograph (GC). (A) A mixture of several compounds is injected into a heated chamber (the injector) where the sample vapor is mixed with a current of inert gas and is swept into the entrance of the column. (B) As the vapor is carried along in the flow of inert gas, compounds with greater affinity for the column lining will travel more slowly and separate from compounds with less affinity for the lining. (C) As compounds continue to move through the column, their separation from one another increases. (D) At the end of the column, each isolated compound exits (“elutes”) sequentially into a detector, which produces a voltage proportional to the amount of the compound passing into it. (E) The voltage output of the detector is plotted against time.          

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.   

 

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Figure 2. A coupled gas chromatograph – electroantennographic detector (GC-EAD). (A) A mixture of several compounds is injected into a heated chamber (the injector) where the sample vapor is mixed with a current of inert gas and is swept into the entrance of the column. (B) As the vapor is carried along in the flow of inert gas, compounds with greater affinity for the column lining will travel more slowly and separate from compounds with less affinity for the lining. (C) The column effluent is split in two, with one half transmitted to a flame ionization detector (FID) and the other half carried to the insect antennal preparation. (D.1) The FID produces a voltage proportional to the amount of organic compound in the column effluent (D.2) Column effluent is mixed with purified air and blown over an insect's antennal attached to an amplifier. (E) The amplified electrical outputs of both the FID and antenna are plotted simultaneously

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.   

 

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                  Figure 3. Example of GC-EAD output. Extract from hindguts of female southern pine beetle, Dendroctonus frontalis , was analyzed on a GC-EAD fitted with an antenna from a male beetle. Three recognized compounds from the hindgut extracts are labeled. Several unstudied (unlabelled) compounds also produced antennal responses and are currently undergoing further study. It is noteworthy that, while trans -verbenol and myrtenol have demonstrated behavioral activity with male southern pine beetle (myrtenol is an inhibitor and trans-verbenol is an attractant synergist), cis -verbenol has not. The FID peaks of these three compounds were identified by matching their retention times to those of commercially-available versions of the same compounds injected into the GC. Identifications were confirmed by re-analyzing the extract with a coupled gas chromatograph-mass spectrometer (GC-MS) using the same column and GC operating parameters as used in the GC-EAD analysis.

Mention of trade names is for information purposes only and does not imply an endorsement by the USDA.  Questions, comments, and corrections are welcome and should be sent to Brian Sullivan at briansullivan@fs.fed.us.