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Pheromone Communication in Moths

Evolution, Behavior, and Application

UNIVERSITY OF CALIFORNIA PRESS

Reminiscence of the Early Days

WENDELL L. ROELOFS

BECOMING AN ENTOMOLOGIST

CHALLENGES TO PHEROMONE IDENTIFICATIONS

Oak leafroller, Archips semiferanus (Tortricidae) European corn borer, Ostrinia nubilalis (Crambidae) Codling moth, Laspeyresia pomonella (Tortricidae) Sex attractants used as a taxonomic tool

CHALLENGES TO BEHAVIORAL STUDIES

Is a sex attractant really an attractant? Are all emitted compounds really pheromone components? Blend versus individual component roles

THE NEXT PHASE

REFERENCES CITED

Becoming an Entomologist

One of the hottest topics at Entomological Society of America meetings (ESA) in the mid-1960s was anything to do with insect pheromones. The recent decoding of the silkworm moth, Bombyx mori (Bombycidae), pheromone by German scientists (Butenandt et al. 1959) after three decades of research showed that it was possible to unravel the mysteries of these mating messages. The term "pheromone" had recently been coined (Karlson and Luscher 1959) to describe these chemical signals, and much discussion was centered on the exact meaning of this new term. A plea in Rachel Carson's book Silent Spring to develop insecticide alternatives also helped to generate funds for research on pheromones and their use in pest monitoring and management programs. The idea for the practical use of pheromones was proposed in 1882 by J. A. Lintner, the first New York state entomologist (Lintner 1882). He had observed the great attraction that female Promethea moths had for conspecific males from long distances and wrote: "Can not chemistry come to the aid of the economic entomologist, in furnishing at moderate cost, the odorous substances needed? Is the imitation of some of the more powerful animal secretions impracticable?" Paper sessions and night discussions at the ESA were packed as the few scientists involved in the pheromone field debated questions regarding pheromones and their practical use. It was my great fortune to come into this scene in 1965 as part of a new thrust by the Entomology Department of Cornell University at the New York State Agricultural Experiment Station in Geneva to develop a research program on pheromones of moth pest species.

Paul Chapman, the Chair of the department, felt that the fastest route into the pheromone field was to hire a chemist. He was a wise man and elicited the help of the renowned chemist at Cornell, Jerry Meinwald, to send out the position statement to his colleagues. My postdoctoral advisor at MIT got the statement and showed it to me. Although I had no training in entomology, I was intrigued by the possibility of conducting research on pheromones and applied for the position and got it. The search committee must have been impressed with my PhD thesis on "Cyclization of ylidenemalononitriles" in Organic Chemistry from Indiana University.

The entomologists at Geneva were eager to collaborate with me and I quickly set up a project on an apple pest, the red-banded leafroller moth, Argyrotaenia velutinana (Tortricidae), which had become resistant to the current pesticides and had become a major pest. Another project, which was funded by the NSF, was on the giant cecropia moth, Hyalophora cecropia (Saturniidae). This species was being mass reared by a fellow faculty member, Frederick Taschenberg, at the Fredonia Research Laboratory and was included in the research since it was a very large insect and thought to be an easy subject for pheromone identification. It turned out that the cecropia moth has very little stored pheromone, and the chemical structure apparently so complex that so far it has eluded all efforts on its identity.

To guide our research there was little information on pheromone structures or how to identify them. After the publication of the silkworm moth pheromone in 1959, Milt Silverstein and Dave Wood reported (Silverstein et al. 1966) on a chemical blend of three compounds for a bark beetle, and then Bob Berger (1966) published the pheromone of the cabbage looper moth, Trichoplusia ni (Noctuidae) as (Z)-7-dodecenyl acetate (Z7-12Ac). With little information available on how to identify pheromones, I decided to take a trip to the USDA labs in Washington, DC, to get up-to-date on methodology since the scientists there had been involved for years with the pheromones of a number of moth species, such as the gypsy moth and pink bollworm moth. However, accumulating thousands of field-collected whole moths in barrels of benzene did not seem like a good pheromone purification scheme. Therefore, we jumped into the fray by setting up a mass rearing effort and clipped 50,000 female abdominal tips in ether or dichloromethane for isolation of the pheromone by column, thin-layer, and gas chromatography. In a couple of years we identified the red-banded leafroller moth pheromone as (Z)-11-tetradecenyl acetate (Z11-14Ac). This monounsaturated acetate was synthesized by my first postdoc, Henry (Heinrich) Arn, using a Wittig reaction, and then tested in apple orchards with baited ice cream carton sticky traps to show its great activity in trapping male moths (Roelofs and Arn 1968).

I was fortunate to associate with faculty colleagues, Paul Chapman and Sieg Lienk, who were preparing a book (Chapman and Lienk 1971) on over 50 leafroller species found in wild apple trees in New York. They collected larvae by beating branches of wild apple trees around Memorial Day and then reared the dislodged larvae for identification. I joined them on these field excursions and this allowed not only for my entrée into the world of entomology, but also for material to start cultures of several pest leafroller species from the collected larvae. We were able to identify the pheromone of a number of these leafroller species and found that many also used Z11-14Ac. Since the female extracts were very specific in attracting conspecific males, we anticipated that specific blends must be involved, similar to what had been previously reported for bark beetles. After more research on the gland extracts and numerous field tests, the postdoc chemists in my lab, Henry Arn, Ada Hill, and Jim Tette, were able to show that these leafrollers used precise ratios of Z/E isomers with various additional components added to make a specific blend for each species. Interestingly (and fortuitously), the redbanded leafroller males were initially captured in great numbers with Z11-14Ac lures because this species uses a 92:8 ratio of Z/E isomers, which is exactly what was produced using the Wittig reaction to synthesize Z11-14Ac.

Challenges to Pheromone Identifications

Oak Leafroller, Archips semiferanus (Tortricidae)

By the mid-1970s species-specific pheromone blends had been documented in numerous moth species. However, Larry Hendry at Penn State published results that led to a proposal of startling new concepts in insect chemical communication and evolutionary biology that seriously challenged the existence of species-specific pheromone blends. He was a bright, young scientist in the Chemistry Department investigating the pheromone of the locally abundant oak leafroller moth. His initial studies on extracts from the pheromone glands indicated activity in gas chromatography (GLC) collections at the retention time of 14-carbon acetates. At that point, they conducted field-trapping studies using large sticky vane traps. These traps were normally used for bark beetles in very dense populations and could intercept thousands of insects, male and female, even with blank (unbaited) traps. If a single trap containing a test chemical captured more males than the 3000+ males caught on a blank trap, the chemical on that trap was concluded to be an attractant for oak leafroller males. Field tests involving various monounsaturated 14carbon acetates throughout the woods in an array of different species of oak trees led to the conclusion that the pheromone used by oak leafroller moths differed among white, red, and black oak trees and could include acetates mainly with Z3, Z4, or Z10 double bonds. This conclusion along with anecdotal observations that male oak leafroller moths were attempting to mate with leaves of the different host trees led to the controversial conclusion that with pheromones, "You Are What You Eat." In other words, the larvae obtained the pheromone from host leaves and the sequestered chemicals could be different for each host species. As a result moth sex pheromones could vary among populations. Analyses of the different oak leaves by the Penn State scientists suggested the presence of the pheromone structures to support this conclusion (Hendry et al. 1975a, 1975b, 1975c).

This conclusion undermined the growing information on pheromone blends since it suggested that pheromone blends could be highly variable within a species, and that pheromones for monitoring and insect control programs would probably fail as the pheromone production and response were plastic. Many leading biologists and chemical ecologists readily accepted the concept of "You Are What You Eat" as a viable hypothesis. With this support, the new idea was widely promoted by the Press, discussed in symposia at national and international ecology meetings, and published in several research papers in Science. Jane Brody, a columnist for The New York Times, later summed up the threats to the pheromone field in an article that discussed the pros and cons of this new idea. She said:

If Dr. Hendry were right, pest control that relied on manmade versions of insect sex attractants, or pheromones, would be doomed, because no one could predict which chemical the insects would respond to. In scores of research projects costing millions of dollars, synthetic pheromones are being used to detect insect invasions by luring them into pheromone-baited traps and to disrupt insect mating patterns by widely spraying the pheromone that ordinarily draws in the male insect to a receptive female. Dr. Hendry's report in April 1975 that at least one insect pest, the oak leafroller, derived its pheromone from leaves it ate and produced different chemicals from different diets threw the field of pheromone research into temporary disarray.

In the mid-1970s, Jim Miller came as a postdoc to our research group after receiving his PhD from the Entomology Department at Penn State. He was appalled by how the field data on the oak leafroller were collected by the scientists in the Chemistry Department and was eager to reveal the fallacies of the new hypothesis. He initiated a rigorous re-investigation of this pheromone. He reared the larvae on a pinto bean diet and showed that the female moths produced a precise ratio of 67:33 E/Z-11-14Ac, obviously without obtaining pheromone from their diet. He then analyzed the pheromone from female moths reared on the different oak leaves and found the same ratio of pheromone components from all oak species. Furthermore, male moths reared on the various leaves were responsive in the lab only to that precise blend of geometric isomers. The final blow to the hypothesis was delivered with data from a large field-trapping study that our research group conducted in the woods near Penn State. Treatments placed in small sticky traps were replicated 10 times, and included an array of E/Z11-14Ac isomeric blends from 0:100 to 100:0, as well as other monounsaturated acetates purported to be pheromones by the Penn state scientists. The results showed that male moths were trapped only with the female-produced blend (70:30) of E and Z11-14Ac, and none by the other suggested monounsaturated acetates or off-blends of the 11-14Ac. Thus, the oak leafroller pheromone was found to be similar to other leafroller pheromones in using a very specific E11/Z11 ratio of chemicals. These data that disproved the hypothesis were presented at a fully packed ESA meeting and published in Science (Miller et al. 1976).

However, the idea did not die easily, since Science then published a rebuttal by Hendry (1976). He attributed the differences in results between the two studies to the use of different techniques and did not retract the hypothesis. Our lab then collaborated with the postdoc and graduate student from Penn State who were involved in the project and had them use procedures developed in our laboratory to verify the pheromone identification. It resulted in a complete retraction in Science (Hindenlang and Wichmann 1977). Hindenlang and Wichmann stated:

Our present results indicate that the earlier data and derived hypotheses should be reconsidered. We do not deem it appropriate to advance a hypothesis regarding a direct association between plant chemistry and insect sex pheromones. Furthermore, we retract previous reports and interpretations of data suggesting such an association ... In our present analysis of the plant material, we found compounds that were clearly not tetradecenyl acetate but gave patterns similar to it.

In other words, the leaves did not contain pheromone compounds. These two brave scientists were under much pressure to maintain their support for the new hypothesis, but in the Jane Brody report in The New York Times they said that as far as they were concerned, the theory of sex pheromones in plants is dead.

European Corn Borer, Ostrinia nubilalis (Crambidae)

Another interesting challenge to a pheromone identification came after Jerry Klun characterized the European corn borer pheromone from 10,000 females (Klun and Brindley 1970) as Z11-14Ac. He conducted the research in collaboration with a Professor of Entomology at Iowa State who was an acknowledged leader in managing European corn borer populations in the Midwest. However, in 1972, a postdoctoral chemist in my group, Jan Kochansky (Kochansky et al. 1975), found that European corn borer females in certain field plots in New York produced and males were specifically attracted to the opposite isomer, E11-14Ac, and not to the Z isomer as found in Iowa. This was a great collaboration that we had with my colleague Chuck Eckenrode and his technician Paul Robbins. We presented these findings in a symposium at an ESA meeting to a packed ballroom audience, but they were not well received by all. The Iowa State collaborator shouted from the middle aisle of the ballroom that it had to be a wrong identification and that a "corn borer, is a corn borer, is a corn borer." The controversy was resolved in the next year by full cooperation on both sides by exchanging insect cultures obtained from Iowa and New York and conducting in-depth analyses of the pheromone of both populations. Both laboratories found that indeed the Iowa population used a 97:3 Z/E blend and the New York population used the opposite 1:99 Z/E blend. This finding provided good evidence for pheromone polymorphism in this species and led to years of research on three genetically different European corn borer "races" in New York labeled bivoltine Z, univoltine Z, and bivoltine E as defined in the field mainly through the efforts of Paul Robbins (Roelofs et al. 1985).

Codling Moth, Laspeyresia pomonella (Tortricidae)

In the years leading to the early 1970s there were many sex pheromones and sex attractants reported to be monounsaturated acetates and alcohols with 12-, 14-, and 16-carbonlength chains (Roelofs and Comeau 1970). A number of apple pests were included in that list, but one glaring omission was that of the codling moth, which is a major worldwide pest of fruit. By 1970, USDA scientists had initiated a project on this species and evidently had already extracted thousands of female moths for pheromone identification. That pheromone presented an interesting challenge so we took it on with the help of a novel technique that was set up in our laboratory by a creative graduate student, André Comeau.

In the 1960s, Dietrich Schneider in Germany was conducting research on antennal responses of male silkworm moths to the newly identified pheromone (Schneider 1962). He developed a technique in which an antenna was connected between two electrodes, and responses to volatiles recorded by measuring the depolarization of the antenna as it responded to active compounds. Comeau set up this electro-antennogram (EAG) in our lab as an analytical tool. He found that the large EAG responses from male antennae to their own pheromone compounds could be used to determine GLC retention times of activity, with effluent collections from injections of crude pheromone-gland extracts. The advantage of collecting the effluent in capillary tubes compared to the later use of the combined GLC-EAD technique was that the active material could be rinsed from the tubes and used for an injection on a GLC column of different polarity or used for micro-reactions for information on the double bond position or compound functionality. The retention times could be used to determine if there were several major active compounds in the crude extract, if they were alcohols or acetates, and the length of their carbon chains.

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Excerpted from Pheromone Communication in Moths by Jeremy D. Allison, Ring T. Cardé. Copyright © 2016 The Regents of the University of California. Excerpted by permission of UNIVERSITY OF CALIFORNIA PRESS.
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