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organic_gardening2.gif  Is Caffeine an Effective Pesticide  organic_gardening3.gif
  Against Drosophila (fruit fly)?  

A Science Fair Research Project - Zoology

This project won first at PJAS Region 1B, first at PJAS States, second at Montgomery County Science Research Competition, and third at Delaware Valley Science Research Competition.

This project contains:
         bullet.jpg  Abstract (a summary of the project)
         bullet.jpg  Literary Research
         bullet.jpg  Problem
         bullet.jpg  Hypothesis
         bullet.jpg  Procedure
         bullet.jpg  Constants
         bullet.jpg  Data
         bullet.jpg  Pictures
         bullet.jpg  Graph Analysis
         bullet.jpg  Conclusions
         bullet.jpg  Extension - Explanation of the spraying data
         bullet.jpg  Application to Home Gardeners
         bullet.jpg  References Sited


bullet.jpg  Abstract/Summary  bullet.jpg

         Caffeine is a chemical found in coffee, tea, cola and chocolate. It is produced naturally by plants. I investigated caffeine for pesticidal properties against drosophila. In my hypothesis I indicated that caffeine would be detrimental to drosophila. I based this claim on my research. Essentially, caffeine can substitute for adenosine – an inhibitory neurotransmitter. Caffeine prevents the "slow down" message of adenosine from being passed between nerve cells and substitutes its own "speed up" message. Overactive neurons have many hormonal ramifications (including the release of adrenaline.) To analyze the effect of caffeine on the flies, I created several different caffeine solutions and mixed them with the food supply. I then placed the flies in their respective containers, and observed them for fatalities, behavior, and life cycle data. The data that I found indicates that caffeine is effective as a pesticide: by day 8, all of the flies in the 10 g/L group and the 20 g/L group were dead. The caffeine postponed the life cycle of the 1 g/L group (the 20 g/L and 10 g/L group did not lay eggs). Additionally, the Time to Settle – a relative measurement of the activity of the flies – was greater for the higher concentrations of caffeine. I concluded that caffeine affected the flies as predicted. The overactive neurons contributed to greater activity in the flies and influenced the hormonal gland of the flies – this was the cause of the delay in the life cycle. The fly fatalities resulted from the failure of overactive organs – these were caused by the release of excessive adrenaline. As an extension to my project, I sprayed caffeine on the flies. The results from spraying were similar to ingestion, leading me to believe that caffeine can successfully penetrate the exoskeleton. For further research I could analyze the practicality of caffeine as a pesticide (Does it kill other harmful insects? Does it break down when in the environment?) I could also take an in depth look at the effect of caffeine on the flies (i.e. potential mutagenic effects, the response to specific stimuli, etc.)

bullet.jpg  Literary Research  bullet.jpg

         Caffeine is a chemical found in coffee, tea, cola, and several other plant products. It is produced naturally by plants. Caffeine, in its various forms, is consumed by millions of people each day; however, caffeine has been shown to have some detrimental effects.

Caffeine and Adenosine have a similar double ring structure

         The source of caffeine's "kick" and it's dangers lie in the molecular similarity between it and adenosine. Adenosine, a primary component of ATP and DNA, fills an important role in the brain and central nervous system. Adenosine belongs to a class of chemicals known as neurotransmitters. Neurotransmitters are the communication pathway between neurons (nerve cells). Every nerve cell is composed of a central body and several long, tentacle-like structures called axons. When the nerve cell receives an impulse, the membrane of the fiber becomes depolarized, and sodium ions move across the membrane into the cell. After the membrane becomes depolarized at one point, the adjacent sections of membrane also become depolarized. Neurotransmitters like adenosine become important when the impulse reaches the end of the fiber, or synapse. A nerve impulse sets off the release of certain neurotransmitters in the synapse. The released neurotransmitters are then received by chemical-specific receptors in the adjacent synapse. These receptors only receive molecules of a certain structure.

The neuron communicates impulses with neurotransmitters

         When few nerve impulses are being communicated, the synapse releases adenosine. Adenosine is an inhibitory neurotransmitter – it tells the neurons not to fire. Additionally, adenosine stimulates the production of other inhibitory neurotransmitters. When caffeine enters an organism, it fits into the adenosine receptors - Not only does caffeine have a similar structure to adenosine, but it is also more nucleophilic and electrophilic and is accepted over adenosine by the adenosine receptors. Caffeine blocks out the "slow down" message of adenosine, while substituting it's own "speed up" message. It stimulates the release of other excitatory neurotransmitters (nor-adrenaline and dopamine) while retarding the release of inhibitory neurotransmitters (GABA and serotonin). Essentially, caffeine spurs the neurons to high levels of activity.
         Additionally, caffeine acts as a phosphodiesterase inhibitor. Phosphodiesterase is an enzyme located in cells. It breaks down the cyclic nucleotides cAMP and cGMP, which "are second messengers that carry signals from the cell surface to proteins within the cell." (http://www.mblab.gla.ac.uk/tubules/map/pde.html) If these messenger molecules are not broken down, they simply continue to deliver their message, again and again, to the proteins within the cell. The result: a stimulus that continues to stimulate without cease. Cyclic nucleotides deliver excitatory messages; therefore, by allowing them to continuously deliver their message, cell activity is increased. Caffeine, which inhibits the production of phosphodiesterase, thereby leaves the cyclic nucleotides free to deliver their excitatory message again and again, and results in an increase in cell activity.
         The consequences of high neuron activity are numerous. The neurons, which are linked to the organs of hormonal release, have many hormonal consequences. Primarily, there is an increase in the amount of adrenaline released. Adrenaline, a hormone that directs cells to release glucose into the blood stream, stimulates a fight of flight reaction in an organism.
         Drosophila, although significantly smaller than humans, have a fairly sophisticated central nervous system and endocrine system. Their brain is connected to a thoracic ganglia (a mass of nerves) by the cervical connective. This complex nervous system means one thing: there are many nerve cells for caffeine to affect. Drosophila has an organ of hormonal release called the corpus allatum. The corpus allatum releases countless hormones, including adrenaline. These hormones have similar effects on drosophila as on humans: in his Insect Hormones, Wigglesworth notes that "adrenaline will produce pharmacological effects on the heart beat, gut movements, etc., in insects." (Wigglesworth, p. 101) Insects have many of the same neurotransmitters as humans: including GABA, dopamine (in high amounts), serotonin (5-HT), and nor-adrenaline. These chemicals perform the same functions in insects as in humans: "Dopamine induces a current in cockroaches [fires the neurons]" (Burrows, p. 185) and "catecholamines…[dopamine and nor-adrenaline] act as synaptic transmitters." (Wigglesworth, p. 101) Like humans, insects have 3 types of neurons: sensory, association, and motor. (Demerec, p. 481) The variety of neurons opens several different functions to the influence of caffeine.

         A message received by the nervous system is transported through the human body in less than a second. The consequence of this rapid communication is that chemicals like caffeine influence the entire body shortly after they are introduced.
         From this research, it is feasible that caffeine would have a detrimental effect on flies and other organisms. Several studies have already been conducted involving the effect of caffeine on flies, rats, etc. One group experimented with the effect of caffeine on the social investigatory behavior of the male Norway rat: "At a dosage of 20 mg/kg [body weight] or greater, caffeine counteracts a decrease in social investigation attributed to copulatory experience. At a 10 mg/kg dosage, caffeine increases social investigatory behavior prior to sexual exposure but has no comparable effect after sexual exposure…[they concluded that] acute caffeine exposure apparently interferes with access or retrieval of reference information in long-term memory." (Thor) Another group experimented with Drosophila: "The productivity of Drosophila prosaltan treated with six concentrations of caffeine (from 50 micrograms/ml [.5mg/L] to 2,500 micrograms/ml [2.5 mg/L] of culture medium) during ten generations (approximately 8 months) decreased in a dosage dependent manner in every generation, but at the end of the treatment the flies in all experiments recovered normal productivity, except for those treated with 2,500 micrograms/mL. Longevity in the tenth generation was significantly reduced in males and females only in the 2,500 micrograms/ml dosage, with males being much more affected than females. In a previous study in which the treatment was done in a single generation, productivity exhibited only a partial recovery when the treatment ceased and longevity was significantly reduced in 1, 500 micrograms/ml dosages. The hypothesis of selection occuring in ten generations leading to recovery in productivity and to a reduction in the processes which cause a decrease in longevity is being considered." (Itoyama) In another experiment, "Parameters of sexual behaviour were studied in Drosophila prosaltans treated with 2,500 micrograms/ml of caffeine per 1 ml of banana culture medium. The mating frequency and copulation duration were greater in control than in treated flies, while the pre-copulation duration was greater in treated flies than in controls." (Itoyama)
         Yet the question remains: Would caffeine be an effective pesticide when sprayed on insects. Research seems to indicate that caffeine would be effective when sprayed. A sprayed pesticide, if effective, would have to also be absorbed by the exoskeleton. "The exoskeleton, integument, or body wall of an insect is a very remarkable structure which furnishes ample protection to the animal from moisture, dryness, disease organisms, certain animal parasites, shocks, temperature, and many other dangers. It is composed of relatively thin, rigid, hard, leathery tough plates joined by thick or thin elastic and often fragile tissues." (Essig) The ability to penetrate the exoskeleton is essential for a new pesticide to be effective. Unfortunately, the exoskeleton is composed of several layers, and most have some impermeability.

         The exoskeleton is composed of three principal layers – cuticula, hypodermis, and basement membrane. The cuticula has a stratified appearance and consists of two distinct layers – an outer cuticula, exocuticula, and an inner cuticula, endocuticula. The exocuticula is covered on the outside by a thin layer about one micron in thickness known as the epicuticula. The most characteristic substance in the exocuticula and endocuticula is chitin. This compound is a nitrogenous polysaccharide and is insoluble and resistant to the action of water, alcohol, and diluted alkalis and acids. The epicuticula is nonchitinous in nature. It is composed of substances that protect insects from excessive dessication, humidity, and disease; thus enabling them to live under a wider range of environmental conditions. The hypodermis consists primarily of a single layer of cells which secretes the cuticula. The basement membrane is a thin, noncellular membrane that forms the inner lining of the hypodermis. (Little)
         Chitin, the substance found in the endocuticle and exocuticle, makes up "20 to 50 per cent" (Essig) of these layers. Chitin is insoluble and impermeable to water and other solutions; therefore, these two thick, chitinous layers make a formidable barrier. The epicuticula is also impermeable, though it does not contain chitin. "The exoskeleton is made of plates or sclerites which are usually hardened or sclerotized." (Little) These hard plates are another significant barrier. The soft membrane in between these plates – sutures – are a potential doorway for pesticides.
         Knowledge about the structure and function of the insect exoskeleton has proven critical in developing insecticide formulations that are able to penetrate this multi-layered protective covering…Knowledge of the nervous system of insects has led to the development of several types of insecticides designed to disrupt normal nerve function. Some of these are effective simply by contacting the insect. (http://www.nyseas.cornell.edu/ent/biocontrol/info/primer.html)
         Some pesticides that affect the insect nervous system are effective simply by contact. It is feasible that a caffeine solution, which affects the nervous system, will be effective on contact. Any influence that a caffeine solution would have on a insect is due to penetration of the exoskeleton: only "insects with chewing mouthparts can be selectively controlled by some insecticides that are applied directly to plant surfaces and are only effective if ingested." (http://www.nyseas.cornell.edu/ent/biocontrol/info/primer.html)

Drosophila Life Cycle          It is also important to know a little about the life cycle of drosophila. "A fresh culture of D. melanogaster will produce new adults in two weeks; eight days in the egg and larval stages, and six days in the pupal stage. The adult fruit flies may live for several weeks…The pupa begins to darken just prior to the emergence of an adult fly." (Flagg)
         When monitoring the flies for the negative effects of caffeine, fatalities will obviously be the primary measurement. Time to settle, however, may also have value. When Drosophila settles, it enters a sleep like state. The time that it takes to enter that state can be effectively measured.
         "To facilitate the genetic study of sleep, we documented that rest behavior in Drosophila melanogaster is a sleep-like state. The animals choose a preferred location, become immobile for periods of up to 157 minutes at a particular time in the circadian day, and are relatively unresponsive to sensory stimuli. Rest is affected by both homeostatic and circadian influences: when rest is prevented, the flies increasingly tend to rest despite stimulation and then exhibit a rest rebound. Drugs acting on a mammalian adenosine receptor alter rest as they do sleep, suggesting conserved neural mechanisms." (Hendricks)

Other Pertinent Information

Caffeine:

    Caffeine is a white powder; at room temperature, 1 gram is soluble in about 60 mL (Sigma)

Insects:

IV.  Transferring the Flies
  1. Obtain the D. Melanogaster culture.
  2. Obtain one of the subculture tubes.
  3. Upend the subculture tube over the tube with the D. Melanogaster culture.
  4. Quickly remove the stoppers of both tubes and connect their open ends.
  5. Allow approximately 10 flies to enter the subculture tube.
  6. Quickly disconnect the tubes and replace the foam stoppers.
  7. Repeat with the other subculture tubes.

V.  Observing the Flies

  1. Place the subcultures in a styrofoam incubator – set the thermostat to 25°C
  2. Observe the flies once every day. Look for:
    • Behavior (especially mating rituals) – refer to Animal Behavior in Laboratory and Field
    • Fly fatalities – count the number of dead and living flies
    • Time to Settle (TTS) – The time that it takes for a majority of the flies in the tube to stop moving after agitating the tube
    • Life cycle observations – Are there any larvae or pupae?
    • Qualitative observations – What do the pupae/larvae/flies look like
    • Height of the culture medium
  3. Allow the flies to go through one full life cycle before terminating observation

 Constants  
  • Temperature
  • Lighting conditions during observations

 Data - Click for the enlarged graph  

Fly Fatalities by Day 8 (caffeine in food)

Average Time to Settle for First 15 days (caffeine in food)

Life Cycle Observations (caffeine in food)

Fly Fatalities by Day 5 - Sprayed with 0.5 mL of Solution

Average Time to Settle by Day 8 - Sprayed with 0.5 mL of Solution

 Pictures - Click to enlarge; hold cursor over picture for a description  

20 g/L and 10 g/L 1 g/L and 1 mg/L: in the 1 g/L group several flies became lodged in the foam stopper 100 mg/L and 10 mg/L
.01 mg/L and .1 mg/L .001 mg/L Control #1 and Control #2

 Graph Analysis  

         The most significant data is the fly fatalities graph. All of the flies in the 20 g/L group were dead by the fourth day, and all of the flies in the 10 g/L group were dead by the eight day. The other fatalities, which were not more than a single fly, are considered insignificant. The fatalities indicate that at concentrations of 10 g/L and 20 g/L, caffeine is fatal to drosophila.
         The life cycle graph brings the influence of caffeine on the reproductive cycle of the fruit fly to the forefront. Note that there is no data for the 10 g/L and 20 g/L groups – the flies in these groups died before they could lay eggs. Analysis of the other groups, however, reveals an interesting trend. The day when the first pupae were spotted is day 7 or day 8 for all the groups except the 1 g/L group. The first pupae in the 1 g/L group appeared two days after the pupae in the other groups. With the exception of the 1 g/L group, the first dark pupae of all the groups were observed on day 11. Obviously, this is the normal time for the first dark pupae to appear. Dark pupae were not observed in the 1 g/L group until three days later – day 14. The first new flies for all of the groups except 1 g/L appeared on day 12 – exactly one day after the first dark pupae. The first new flies in the 1 g/L group were spotted three days later – day 15. From the analysis of this graph, it becomes obvious that in a concentration of 1 g/L, caffeine delays the reproductive cycle of the fruit fly.
         The average time to settle graph also reveals conclusive data on the influence of caffeine. But first, it is important to reiterate that Time to Settle (TTS) has value as a relative measurement – it gives accurate comparisons between the groups of flies. If Group A takes longer to settle than Group B, then Group A is definitely more active than Group B. According to the graph, flies that were treated with 1 g/L and 100 mg/L took longer to settle than flies treated with lower concentrations of caffeine. The caffeine made these groups more active than the groups with lower caffeine concentrations. The 1 mg/L group was also much more active than the groups with lower caffeine concentrations. It is important to note, however, that the 10 mg/L group was far less active than it's neighboring groups. We may conclude from this data that the influence of caffeine on the motion of drosophila becomes insignificant at concentrations of 10 mg/L and lower. The other explanation is that the 10 mg/L or the 1 mg/L time to settle values were inaccurate. Considering that the time to settle values were all relative to the other tubes and that the relative comparisons were highly accurate, it seems illogical that the "blip" in the graph at 10 mg/L is the product of experimental error.

 Conclusions  

         Caffeine most definitely has a negative effect on drosophila. Caffeine accelerates the firing of neurons by blocking adenosine from the synapses and acting as an artificial excitatory neurotransmitter. The caffeine caused fatalities by overburdening the heart, stomach, and other organs of the flies. As adrenaline surged through the systems of the flies, their organs shifted into high gear. The constant influx of caffeine kept a continuous strain on their bodies. Eventually, their organs became exhausted and failed.
         In the 1 g/L group, there was not enough caffeine to cause fatalities. The caffeine did produce tangible results, however. It slowed down the reproductive cycle of the flies significantly. This development was probably due to the influence of caffeine on the "ring gland." The ring gland is an organ of hormonal release in drosophila – it releases the hormones that initiate and conclude the phases of drosophila's life cycle. The ring gland is influenced by neuron activity; therefore, overactive neurons could result in complications with hormone release and problems with the reproductive cycle. Other experiments with drosophila and caffeine produced results similar to this experiment: Scientists found that the flies exposed to caffeine experienced a reduction in mating frequency and copulation duration and an increase in pre-copulation duration (the time before the flies mated). These findings would contribute to a slower reproductive cycle. Overactive neurons influenced the flies in other ways. The excessive neuron firing exaggerated the central nervous system's response to common environmental stimuli. This "exaggeration" became clear when analyzing the response of drosophila to gravity. Drosophila instinctively flee the Earth's gravity – when a culture of drosophila stands on its end, the flies travel to the top of the container. In the 1g/L culture, however, the overactive neurons incessantly drove the flies upward. The overactive nervous system placed an extremely heavy emphasis on the gravitational stimuli, an emphasis so extreme that the flies suffocated themselves in the foam stoppers while responding to it.
         The Time to Settle statistics revealed another interesting effect of caffeine. Caffeine, when administered in high enough concentrations, caused increased motion in the flies. The increase in activity in the flies was probably caused by overactive neurons. Drosophila is stimulated by light, and because the flies were timed under a fairly strong lamp, another "exaggeration" of environmental stimuli probably occurred. There is another explanation for the increased activity. In experiments with male Norway rats, scientists found that caffeine (when given at a high enough dosage) served to counteract a decrease in social investigation that was attributed to copulatory experience. Basically, caffeine spurred the rats to interact with other rats after they had mated (normally, social investigation would be reduced by intercourse.) Though rats and flies are two very different organisms, a comparison may be drawn. The caffeine could have spurred the flies to increased social interaction before or after copulatory experience. Increased social investigation would result in increased motion, which would produce a higher Time to Settle.
         In answer to my hypothesis: I believe that caffeine could be an effective pesticide against drosophila when applied in high enough concentrations. It is clear that in high concentrations, caffeine kills insects. Additionally, caffeine has many advantages over other pesticides. It is produced naturally by plants and is not as hazardous as artificial pesticides. Caffeine can be obtained in large quantities from coffee, tea, and cacao; it would be fairly inexpensive to produce. Additionally, caffeine would quickly break down when introduced to the environment; traditional pesticides would take much longer to biodegrade. This experiment has given insight into the purpose that caffeine serves in plants. Considering that the influence of caffeine on drosophila was entirely negative, plants probably produce caffeine as a natural defense against insect predators.

 Extension  

         After the initial phase of the project, in which I determined whether caffeine was fatal to drosophila when ingested, I was still curious. I wanted to know if caffeine would be absorbed through the exoskeleton - if it would be an effective pesticide when sprayed. To find out, I started a new phase of experimentation. I cultured three new groups of flies. For the control, I sprayed the flies with 0.5 mL of distilled water. For the 10 g/L and 20 g/L groups, I sprayed the flies with 0.5 mL of their respective solutions. The spraying was done with a typical commercial spray bottle and was uniform.
         The data (see following pages) from the spraying experimentation corresponded to the ingestion experimentation. In the 10 g/L group, 75% of the flies were dead by day 5. In the 20 g/L group, 50 % of the flies were dead by day 5 (Note: unfortunately, I was unable to get an equal number of flies in each container. 7 of 14 flies died in the 20 g/L group, while 6 of 8 flies died in the 10 g/L group.) None of the control flies were dead by that time. Flies eat by sponging off their food – pesticides, when sprayed on the surface of their food supply, are usually ineffective. Therefore, the success of the spraying can only be attributed to penetration of the exoskeleton by caffeine.
         The time to settle information also agreed with my expectations. I experimented with a new method of detecting activity: Relative Time to Settle. The first group to settle was assigned a value of one, the second, two, the third, three. I concealed the labels of the groups and took data several times. Every time, the data on relative activity were identical (therefore, I considered the measurement highly reliable.) For the first eight days, the data is very intriguing. On each of those days, the Relative Time to Settle values were in the same order: The control was the first to settle, then the 10 g/L, then the 20 g/L. There was no deviation. Therefore, the average values for that period were 1 for the control, 2 for the 10 g/L, and 3 for the 20 g/L. This dramatic data can only mean one thing: the caffeine penetrated the exoskeleton and had a direct influence on the firing of neurons, and on the level of activity. After this eight day period, the influence of the caffeine on the activity level seemed to diminish.

  Applications to Home Gardeners 

     Any home applications of this science fair project would be experimental. You could make a diluted coffee/tea/cola solution and spray it on detrimental insects. The solution would probably be most effective against smaller insects (i.e. aphids), which would be most susceptible to the caffeine. You might also try ordering some anhydrous powdered caffeine and diluting it to the same concentrations that I used (NOTE - Be careful, powdered caffeine is harmful if inhaled). More importantly, however, my findings on caffeine could lead to the development of powerful, natural pesticides. These could be used on commercial farms and in home gardens.

References Cited
  • Burrows, Malcolm. The Neurobiology of an Insect Brain. Oxford: Oxford University Press, 1996.

  • Chatterjee, Camille. "Coffee: Can it kill?" Psychology Today. Nov/Dec 1999.

  • Cherniske, Stephen. Caffeine Blues. Warner Books, 1998.

  • Demerec, M., ed. Biology of Drosophila. New York: Hafner Publishing Company, 1965.

  • Essig, E. O. College Entomology. New York: The Macmillan Company, 1942.

  • Flagg, Raymond O. Carolina Drosophila Manual. North Carolina: Carolina Biological Supply Company, 1988.

  • Hocking, Brian. Six-Legged Science. Massachusetts: Schenkman Publishing Co., Inc., 1968.

  • Itoyama, M.M. "Effects of caffeine on mating frequency and pre-copulation and copulation durations in Drosophila prosaltans." Cytobios, 1995.

  • Itoyama, M.M. "The development of resistance to caffeine in Drosophila prosaltans: productivity and longevity after ten generations of treatment." Cytobios, 1998.

  • Little, V. A. General and Applied Entomology. New York: Harper and Brothers Publishers, 1957.

  • Manning, Gerard. "A quick and simple introduction to Drosophila Melanogaster." Available http://www.ceolas.org/fly/intro.html

  • "Phosphodiesterase." Available http://www.mblab.gla.ac.uk/tubules/map/pde.html

  • "Product Information Sheet for CO750 CAFFEINE (ANHYDROUS)." Available http://www.sigma.sial.com/sigma/proddata/c0750.htm

  • Ross, Herbert H. A Textbook of Entomology. New York: John Wiley and Sons, Inc., 1967.

  • Shapton, David. Internet Project on Caffeine. Available http://tiger.chm.bris.ac.uk/cm1/DaveS/Welcome.htm

  • Thor, D.H. "Caffeine and copulatory experience: interaactive effects on social investigatory behavior." Physiological Behavior, 1986.

  • Weeden, et al., eds. "Insect Biology and Ecology: A Primer." Cornell University. Available http://www.nysaes.cornell.edu/ent/biocontrol/info/primer.html

  • Wigglesworth, V.B. Insect Hormones. San Francisco: W.H. Freeman and Company, 1970.


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