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Topic 12. Insect Pheromone Response.

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(Name)

I. MATERIALS.

Moths of any species (pink bollworm is especially suitable).

Incubators with reverse light cycle, such as a small Percival chamber. If moths are obtained as pupae, they can be separated by sex and kept separate to ensure males have not mated previously. Males of any age are suitable, but females usually start calling a few days after eclosion.

Pheromone obtained from commercial sources for the experimental moth. (Keep sealed and refrigerated in a separate room until needed.)

A miniature wind tunnel (can be as small as a box one yard long by one square foot in cross-sectional area with a small fan to pull air from one end and a baffle at the opposite end to allow air to enter. Remember, air that is exhausted from the wind tunnel will contaminate the room if allowed to circulate, so use the system wisely, or vent to an adjacent hood). As an alternative to a wind tunnel, the mating behavior of males can be observed when place in Petri dishes with the pheromone source (with appropriate changes to results expected below). Males will only respond if their biological clocks read 2 AM.

Culture two batches of adults, one on a reverse light cycle, the other in sync with the regular light cycle. If you are rearing colonies, be sure to use long day length 14:10; and warm temperatures, at least 30°C or the last instar larvae might go into diapause.

II. LEARNING OBJECTIVES.

Each student should be able to:

Describe the relationship of insect pheromones and mating.

Appreciate the circadian control of mating propensity.

How:

Observing mating behavior of PBW males in the presence of the pheromone or females.

III. INTRODUCTION.

Most animals use chemical odor cues to control their own and the behavior of different of the same species. One of the most important uses of odor cues in insects is the use of chemicals widely known as pheromones emitted by female insects to attract males of the same species for mating. The best known and most exploited pheromones are those of moths. These have been identified, synthesized, often as blends of structures, and manufactured into lures that attract males adults to traps overnight. Use of pheromones has revolutionized the monitoring of sexually mature male adult pest insects, especially Noctuid or other night flying moths. Often pheromone trap catches can detect an early infestation by pest insects at extremely low population densities allowing plenty of time to mount countermeasures.

A great deal is now known about the synthesis of pheromones, their release and detection by highly specialized male antennal receptors. In addition, we now know that insect have a rich behavioral repertoire that accompanies the use of pheromone lures for mating. For example, we know that most male insects cycle into receptivity to the female pheromone only at certain times during the night, and females cycle into a synthesis and release period that closely coincides with the same window in time. Thus the mating behavior of insects can be highly regulated.

The cycling into receptivity by these night-flying insects is one of the marvels of nature. Only when "conditions" are favorable do the females emit and males respond to the mating cues. The conditions necessary to trigger these behaviors have been revealed to include temperature and wind conditions within certain well-defined limits, and also the presence of host plants susceptible to oviposition by gravid females. When these conditions are not present, the adult moths will wait or migrate to other locations before attempting to mate.

IV. DIRECTIONS.

The pink bollworm, Pectinophora gossypiella (Saunders), (or other Lepidopteran), will be provided by the TA in appropriate containers. The moths will have been sexed into separate male and female containers and kept completely separate. The adults will be three days old and mature enough to mate. It is important that the two sexes be kept separate after pupation and the males not allowed to have perceived the female pheromone while in culture.

A wind tunnel will be set up in the demonstration room.

Before the demonstration begins, a short lecture will be given based on the paper: Schouest, L. P. and T. A. Miller (1994) Automated pheromone traps show male pink bollworm mating response is dependent on weather conditions. J. Econ. Entomol. 87(4): 965-974. This paper reported use an automated device to record pheromone trap entry by male pink bollworm adults in a cotton field south of Blythe, CA during the cotton growing season of 1991. Figure 6, page 972 from this paper shows the extreme dependency of male pheromone trap entry on temperatures being 65^oF (about 18^oC) or above at the time of responsiveness which is in the early morning hours and lasts for only about two hours around 2 to 3 a.m. depending on the exact time of year. Figure 3, page 969 from the same paper shows that wind above a threshold level will also shut down trap catch on any given night. Notice the precise peaks of trap entry activity revealed by the automated device.

Bring two groups of adult moths into the demonstration room in containers that have transparent tops. Note the overall activity of the two different groups. One group has been reared an a reverse light cycle, the other group on a regular light cycle. It is not necessary to dim the lights in the room during this demonstration.

Describe the overall behavior of the two groups of moths in their respective containers here. Try not to touch or otherwise disturb the containers. It might be best to place them inside a glass top observation container with enough room for several students to see.

A. Behavior of male moths reared on a regular light cycle.

B. Behavior of male moths reared on a reverse light cycle.

Now (after the initial behaviors of the moths in the containers is documented) place a pheromone source in the wind tunnel at the upstream end and place the container with the adults being reared on the regular light cycle at the downstream end. Remove the top. It is important to be gentle with the container so as to disturb the adults as little as possible. The TA should handle the moths.

It would also be best to place the transparent sides of the wind tunnel in such a position that will afford the most efficient access by the students. Unfortunately, it is best to get fairly close to the container, so it should be at eye level. Remove the lid, start a stop watch and count the number of adult males that fly to the pheromone source for five-ten minutes.

C. Behavior of regular cycle moths in wind tunnel.

adults released: ____; No. at pheromone source: ____; Time period: ______.

After the proscribed time period is over and data is entered. remove the regular light cycle moths from the wind tunnel by vacuuming.

Now repeat the process using the reverse light cycle males.

D. Behavior of reverse light cycle moths in the wind tunnel.

No. adults released: ____; No. at pheromone source: ____; Time period:________

After the same time period used in the original observation, write down the cumulative total number of adults that visited the pheromone source. If a large number of adults is used, it might be difficult to keep track of the adults. If this is a problem, then have one student call out the elapsed time on the stop watch, and have several students assigned to count the males touching the pheromone source. Several other students, one for each counter, can keep the running tally in blocks of ten or fifteen seconds. These data can be graphed:

Number of adult male moths touching the pheromone

source in the wind tunnel during intervals of

time following release in the tunnel:

Cumulative

[Graph soon to come]

After this experiment is over, leave the reverse light cycle males in the wind tunnel and remove the pheromone source from the wind tunnel. (This might most conveniently be accomplished by introducing the pheromone source into the wind tunnel in a disposable cylinder screened away from the interior of the wind tunnel in such a manner that removing the pheromone source will not leave any residue on the wind tunnel structures.

Once the pheromone is removed, note the behavior of the males left in the container.

After another ten minutes, introduce a container of the reverse light cycle virgin females to the container near the end opposite where the pheromone source was and turn off the fan. Open the container and observe the results. Be careful to note the posture of the males during courtship, and describe what you see at the tip of their abdomens:__________________________________________

Describe the mating behavior of these mating pairs. This will best be done by isolating on one pair. Try to describe the mating behavior from beginning to copulation. Note how long the attraction and pre-copulation takes by glancing at your watch throughout the courtship.

E. Mating behavior of reverse light cycle males and females.

V. SUMMARY ANALYSIS.

Describe what was learned from this exercise.

VIII. REFERENCES.

Schouest, L. P. and T. A. Miller (1994) Automated pheromone traps show male pink bollworm mating response is dependent on weather conditions. J. Econ. Entomol. 87(4): 965-974.

Evans, H. E. 1984. Insect Biology, A Textbook of Entomology, Addison-Wesley Wesley, Reading, MA. See especially Ch. 5, Pheromones and allomones, by G. M. Happ, pp. 114-140.

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(Name)

Appendices