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6. ENTOMOLOGY

1

This is an excerpt from the project "The Effects of Tempera­ture on the Toxicity of Insecticides," presented by finalist Bill Boyer of Fayetteville, Arkansas, at the Ninth National Science Fair.

"In the investigation for this project I exposed insects to definite dosages of insecticides for twelve hours to determine if varied temperatures would effect the toxicity of the insecti­cides used.

The method I used was relatively simple. I took very con­centrated insecticides and diluted them in two or three steps. In the first two of these steps I diluted them with water. In the last step I diluted with a solution of 1 part Karo and 2 parts water. (I used ordinary Karo syrup from the store.) The Karo was put in the solution as food for the flies. I added 1 ml. of the insecticide, Karo and water solution to a 60-ml. glass vial containing 1.5 gm. of powdered clay. The powdered clay was to give the mixture consistency or texture. I used dilutions of insecticide at such rates as ½part of insecticide to a million parts of Karo-water solution, 2 parts per million, and 40 parts per million. I had to test many different rates to find the right one to give a good pattern of kill. For instance, too much insecticide would kill all of the insects and too little would not kill any.

I stirred the clay and the insecticide solution with a glass stirring rod until it made a paste. Then I spread the paste on the wall of the vial, about halfway up. I applied the paste to the wall to give it more surface area.

I then used an aspirator, which is a common device for sucking insects into vials, and put ten fruit flies into each vial. I used fruit flies, for they are (1) standard testing animals, (2) easy to rear in great numbers, (3) easy to handle and (4) easily obtainable.

I took the vials with the flies in them, corked them up and put them in a controlled temperature for twelve hours. The temperature chambers were thermostatically controlled. At the end of twelve hours I counted the number of dead flies.

Aspirator, device for putting insects into vials, and illus­tration of insecticide, clay and Karo-water solution in vial recorded the results. Death was defined as occurring when all visible motion had ceased or when they were unable to right themselves after being placed on their backs. I used chambers with temperatures of 50°, 60°, 70° and 80°F. I had a 90° box available but my controlled check indicated that a 90°

heat without insecticide killed 95% of the insects. There­fore it was not used in the other experiments.

KEY

1. 60 ml. glass vial
2. Cotton to keep insects from escaping
   
3. Fruit flies
   

4. Aspirator—consists of two-hole rubber stopper, glass tubing, rubber tubing

5. Mouthpiece for "sucking" up insects

6. 60 ml. glass vial used in temperature chambers

7. Insecticide, clay and Karo-water solution on side of vial

8. Cheesecloth for keeping insects out of mouth

I ran tests on four insecticides. In each test I had four vials for each temperature. I replicated each test four times. The results have been based upon the study of 160 insects. The graphs that are included illustrate the exact figures of the kill. But the general trend shows that the higher the temperature, the more the kill, with the exception of DDT, which works the opposite—the higher the temperature, the less the kill."

2

"A Study of the Firefly and the Chemical Nature of Its Bioluminescence" was started by Leland N. Edmunds, Jr., of North Miama, Florida, a year or so before he entered the Fifteenth Science Talent Search. Lee has recently graduated from college and is looking forward to graduate work in entomology. His paper on the relation between temperature and the flashing intervals of the adult male firefly was pre­sented at the annual meeting of the Entomological Society of America in December, 1959. Ever since Lee was judged one of the top forty winners in the Fifteenth Science Talent Search, he has spent his summers working with the entomol­ogists of the Agricultural Research Service of the Department of Agriculture.

Part of his Search paper follows.

"For a long time I have been curious to know exactly what produces the lightning bug's light, and in the past year I have done extensive research on this subject. By reading widely, I became familiar with the general characteristics of the firefly, but I was unable to obtain any specific details on the chemical nature of its bioluminescence. With this in mind, I wrote Dr. William D. McElroy of Johns Hopkins Univer­sity, who is experimenting with the firefly, asking him for any information pertaining to this topic. He sent several pam­phlets and some adenosine triphosphate (ATP) and said that it would be very convenient if someone could discover a method for growing these organisms in the laboratory. He also suggested, as a project, the collection of eggs from females onto various media to determine whether it is possible to raise these eggs into larval forms.

I decided to divide my project into two sections: (1) attempts to raise fireflies and related experiments; and (2) study of the biochemistry of the firefly's luminescence. I will present my report under these two headings.

Attempts to Raise Fireflies and Related Experiments

My first step was to obtain the fireflies, and I immediately encountered an obstacle—there was a scarcity of fireflies in my region this year. Their season is supposed to last from June through August, but the first one I saw appeared in late August and the last one on October 6!

I usually hunted the insects with a net in a large field near my house from about 6:30 to 7:30 p. m., and I was fairly successful. Enclosed is a chart indicating the number of fireflies I caught each night, along with other details.

While catching fireflies I was able to observe many other things firsthand, some of which verified what I had read previ­ously. I discovered that as the evening progresses, the fireflies rise to high altitudes, a phenomenon which, to the best of my knowledge, has not been explained.

I also learned much about the firefly's romantic habits, for the flash of light is a signal between the sexes. The female climbs to the top of a blade of grass or a shrub and flashes her light to attract a male. The males fly slowly along near the ground, looking for the females and flashing on the average of once every 5.8 seconds. The female signals back every 2.1 seconds, and thus the male is guided toward her and finally mates with her. I determined these intervals of response with a stop watch. To do this, it was necessary to take numerous readings and find an average, for the interval is directly proportional to the temperature, decreasing on hot evenings and increasing if the night is chillier. I tried to find this exact relation, but I was unable to make enough readings to determine anything conclusive.

I had read that it was possible to decoy the males by placing a flashlight in the grass and signaling with the correct interval of response. I tried this but only was able to make the males hesitate in their flight. However, I had greater success in decoying the females. I took a flashlight, covered it partially with my hand and flashed it suddenly in the characteristic check-mark pattern of the male, about every 5.5 seconds. After three trials, a female answered me from about four yards away, and I tracked her down.

Measuring the time intervals also helped me to identify the species common to my locality as Photinus pyralis, which is the only common eastern firefly with these intervals of response, since nature has developed a specific rhythm for each species.

Also, I determined that the average life span of my fireflies, all of which were subjected to the same abnormal conditions, was 5.4 days.

The fireflies I caught I placed in various modified ter-rariums, hoping that they would mate and lay eggs.

On September 17, I managed to secure a pair and put them in a separate container. They copulated for forty-five minutes, but no eggs were produced, and the female died soon after. Other attempts to get males and females to mate failed. Then, on September 24, I discovered a pair beginning to copulate in the field. I carefully removed them to a jar, and they mated for one and a half hours. . . .

On September 27 I discovered nine yellowish-white true spherical eggs on the leaves of the plant in the terrarium and on the container's sides. Nine more eggs were laid on Sep­tember 29, making a total of eighteen. I made a slide of two, and the rest I placed on a plant in another terrarium and on a piece of moist wood. These were kept at about 65°F. Some of the eggs shriveled up, but by December 10 I still had five of them left, and still unhatched. Then, a rat knocked over the terrarium, and my hopes were destroyed. However, I am planning to try again next year to raise fireflies.

Meanwhile, the last of my other fireflies had died on Octo­ber 14, bringing to a close this part of my project.

Study of the Biochemistry of the Firefly's Luminescence

I have just started working on this part of my project, as most of my time has been spent collecting the fireflies for it as outlined in the preceding part. Briefly, the firefly's light is produced by the oxidation of a substance called luciferin in the presence of a catalyst, luciferase. However, only one flash could be obtained unless there was present adenosine triphosphate (ATP), a high-energy phosphate compound which reduces the oxyluciferin to luciferin again after each flash. These reactions can be described by the following equation: Luciferin + Luciferase + Mg + ATP + O2 →Light -+ Oxyluciferin + Etc.

I dissected the tails from the fireflies I caught, ground several of them up and added a solution of ATP in an effort to produce light. However, this was unsuccessful, even when I added Mg. In the latter experiment, the tails changed from bright yellow in color to a dull brownish-yellow. I can think of at least two reasons for my failure: (1) The tails have become inactive; (2) I did not use enough lanterns. The latter is almost insurmountable, for I cannot use any more as I have only 17 mg. of firefly lanterns altogether.

If I can somehow obtain some more fireflies, I plan to extract and purify the luciferase and luciferin in their tails. However, in the procedure outlined by Dr. McElroy (see Diagrams #1and #2)5 gm. of lanterns are required. I also hope to test for the presence of ATP in various tissues with ground-up firefly lanterns. Another spectacular experiment is to make one solution of luciferin and one of luciferase (luci­ferase is unstable in hot water and thus luciferin could easily be obtained) and pour them together, thus producing liquid light.

Hence, future progress depends on my securing more firefly tails. I have just written to Dr. McElroy on this matter, asking his advice and inquiring whether preserved fireflies from biological supply houses would serve the purpose. In any event, however, I plan to continue my work on the firefly throughout the coming year."

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