Throughout the 1960s and 1970s technical improvements dramatically quickened the pace and productivity of pheromone research. Among the improvements was the use of three techniques known as gas chromatography, mass spectrometry, and nuclear magnetic resonance. These techniques were used in combination with the electroantennogram (EAG). Gas chromatography is a technique for separating components in a vapor based on how quickly they travel through a column containing an absorbent material. Mass and nuclear magnetic resonance spectrometers are used to identify chemical compositions.

In 1970, several groups of researchers were working on identifying the pheromone of the codling moth, an apple orchard pest. Despite a massive effort to analyze the contents of around half a million glands from female moths, the pheromone remained elusive. Then in 1971, Wendell Roelofs and his colleagues at Cornell University made the identification by taking a novel shortcut. First, using gas chromatography, they separated extracts from the glands into fractions. They then tested each fraction with the EAG to determine which fraction contained the pheromone. At this point researchers would typically use spectral analysis to isolate the pheromone in the fraction, a slow and labor-intensive process. Roelofs and his colleagues sped up this step by testing a library of all possible mono-unsaturated compounds related to known pheromones. They used the EAG of the codling moth antenna to test each of the chemicals in the library. As they got closer to the actual structure the EAG response increased, peaking in response to two compounds, each containing a different double bond. This information led to the correct prediction that the pheromone compound contained both double bonds in one compound. When the Cornell team announced its identification of the codling moth pheromone, the news was met with disbelief. Only after the results were confirmed using conventional methods did the new approach gain acceptance.

All these techniques, and others, were used in various combinations. Gas chromatography was linked to mass spectrometry so researchers could both separate and identify the pheromone components in their mixtures. By coupling gas chromatography to the EAG, researchers could detect which components in their insect preparations prompted an electrical response. And the development of capillary gas chromatography allowed researchers to separate compounds that could not be resolved by previous methods.

Along with the physical tests researchers now needed new behavioral assays to determine which chemicals were actually part of the pheromone signal. In the 1930s, English zoologist John Kennedy had developed a special wind tunnel to study how insects orient and move upwind. By the 1970s, Kennedy became curious as to how insects track a sex pheromone back to its source. He used his wind tunnel—a clear plastic tube in which an odor is released at one end and blown through the tunnel by a fan. He knew from previous work on the yellow fever mosquito that flying insects use visual cues for guidance as they follow the trail of an attractant. Kennedy therefore equipped the tunnel with a moving patterned floor to simulate the changing territory beneath the insects’ flight path. He found that moths use the same visual information when tracking pheromones.

Since the 1970s, Kennedy’s wind tunnel and similar devices have proven invaluable to researchers trying to test candidate pheromones. If an insect is stimulated to fly upwind in the tunnel toward the chemical scent, then researchers usually conclude that the scent is indeed a pheromone. The wind tunnel also allows researchers to test various mixtures of chemicals at different release rates to find the optimum lure for field traps.

Thanks to a combination of all of these techniques the quantities of insects that researchers need to pinpoint new pheromones has dropped dramatically.