Experimental Design

Effects of Short Duration Microgravity
On Drosophila Melanogaster (Fruit Fly) Activity

Mark S. Miller and Tony S. Keller

Department of Mechanical Engineering
University of Vermont, Burlington, Vermont USA

**Introduction**
The objective of this study was to determine the effects of short-term microgravity on Drosophila melanogaster (fruit fly) activity. Previous investigator’s experiments on Cosmos satellites and Space Shuttle missions have shown a significant decrease in the life span of male fruit flies after microgravity exposure [1,3,4,5]. Understanding the mechanism(s) behind this reduced life span could lead to important advances in the understanding of the aging process. The increased aging was hypothesized to be induced by an increased locomotor activity, driven by the Drosophila’s negative geotaxic response. This response is the tendency of the flies, when stimulated, to walk in the opposite direction of Earth’s gravitational vector. During microgravity exposure, Drosophila may become more active since they are confused by the lack of gravity and begin searching for the gravity vector. In order to measure the activity of fruit flies a new system based on infrared (IR) emitters and detectors was designed and built. The IR system monitored 240 flies, all housed in linear tracks, using 480 pairs of emitters and detectors. Locomotor activity was determined by counting the number of times that an infrared beam was broken per time period.

**Experimental Methods**
Short-term microgravity was provided by a two stage Nike-Orion sounding rocket launched from Wallops Island, Virginia on June 20, 2000 and a KC-135 flight from Houston, Texas on March 10, 2000. The data presented focuses on three variables, sex (male vs. female), orientation of housing (horizontal vs. vertical), and gravitational level (1-g vs. microgravity). Data was removed if no activity was recorded for the fly throughout the entire experiment. Statistical analysis was performed to determine if significant differences (p < 0.05) existed between the different variables. Besides fly activity, acceleration, pressure, and temperature measurements were recorded as well as video of the fruit flies.

**Diagram of a single chamber in the infrared (IR) monitoring system.**

The Fly Tracks section holds 10 individual flies in separate tracks that are 55 x 2.5 x 4.5 mm each. These tracks allow the plenty of room to move, but constrain the flies to break the infrared beams. The Food Chambers section contains two food chambers at either end of each track to avoid preferentially attracting flies to either end. The housings also contain small air holes to allow access to the ambient atmosphere. The Fly Tracks and Food Chambers sections are sandwiched together creating an inescapable housing and are surrounded by IR emitters and detectors. Two infrared emitter and detector pairs, 30 mm apart, cross each linear Drosophila track. In other words, each individual fly has two emitter/detector pairs to measure activity.

**Diagrams of towers in the infrared (IR) monitoring system.**

Six single chambers are assembled into each tower. Each chamber contains 10 flies, so a single tower holds 60 flies. Both the Nike-Orion and KC-135 flights used four towers, two in the horizontal position and two in the vertical position. Thus, a total of 240 flies were examined during each of these experiments.

**Photo of infrared (IR) monitoring system data collection unit.**

The data collection unit takes the crossing information from 480 pairs of IR emitters and detectors and stores the data in its 16 Mbytes of memory. The sampling rate can be varied from 1 to 16 Hz. The Nike-Orion data was collected at 16 Hz and the KC-135 data was collected at 4 Hz.

**Photos of Nike-Orion and KC-135 integrated payloads.**

The IR monitoring system along with other equipment, including accelerometers, pressure sensors, temperature probes, and a video camera were mounted to the payload structure (middle photo). The payload’s lower portion was covered with a thin aluminum sheet and the upper portion with a metal screen for the KC-135 flight (far right photo). The payload was enclosed in an aluminum skin with a nose cone on top for the Nike-Orion sounding rocket flight (far left photo).

**Nike-Orion Sounding Rocket Data**
	The fruit flies were 2-3 days old at launch and experienced a temperature change of 24.5 – 26.0° C from data initiation until the end of microgravity. During launch the fruit flies experienced maximum accelerations of 20 g’s in the axial direction, and due to spinning for stability, 7 g’s in the radial and 4 g’s in the tangential directions.
The male fruit fly activity data (Figure 1) shows a significant increase in activity between the pad (1 g) and all the microgravity data, except for the horizontally oriented group in the first minute and the final minute of microgravity. During the first minute of microgravity, the horizontal oriented group was significant less than the pad data and statistically different from the vertically oriented group. The decrease in activity was most likely due to the large tangential and radial accelerations due to despining the rocket immediately prior to entering microgravity. These accelerations would have a greater effect on horizontally oriented Drosophila.		Figure 1. Activity level for male fruit flies during segments of the rocket flight. Error bars indicate standard error. The fourth minute of microgravity data lasts only 45 seconds, due to re-entry into Earth’s atmosphere.
The female fruit fly activity data (Figure 2) shows a significant increase activity between the pad (1 g) and all the microgravity data, except for the horizontally oriented group in the first minute of microgravity. As with the males, this decrease in activity was probably due to the despin accelerations.
The male and female activity patterns are similar in that activity greatly increases from the first to the second minute of microgravity and then maintains that level or decreases slightly. Interestingly, neither males nor females show differences between the pad and launch groups, although the gravitational level is changing drastically during the launch phase. The only statistical difference between the male and female data occurs for the horizontally oriented group while on the pad.		Figure 2. Activity level for female fruit flies during segments of the rocket flight. Error bars indicate standard error. The fourth minute of microgravity data lasts only 45 seconds, due to re-entry into Earth’s atmosphere.

**KC-135 Flight Data**
	The fruit flies were 1-2 days old during the flight and experienced a temperature change of 18.0 – 20.0° C from data initiation until landing. The KC-135 flies parabolic maneuvers that produce an alternating 1.8 g, then microgravity (20-25 seconds) and back to 1.8 g pattern. Thirty microgravity parabolas were performed during the flight.
The male fruit fly activity data (Figure 3) shows a significant increase in activity after 10 parabolas for the vertically oriented group, which continues until the parabolas end, and after 20 parabolas for the horizontally oriented group. For each of the microgravity segments, the activity level between the horizontal and vertical groups was significantly different. However, the activity level between these two position groups was not different during level flight before or after the parabolas.		Figure 3. Activity level for male fruit flies during segments of the KC-135 flight. Error bars indicate standard error. Level flight before and after lasted 9.6 minutes and 24.6 minutes, respectively. Each parabola lasts 20-25 seconds.
The female fruit fly activity data (Figure 4) shows a significant increase in activity after parabolas begin for the vertically oriented group, which continues and increases for the duration of the flight. However, a significant increase in activity was not observed until after 20 parabolas for the horizontally oriented group. As with the males, the activity level between the horizontal and vertical groups is statistically different during microgravity, but not during level flight either before or after the parabolas.
The male and female activity patterns are similar in that the activity level increases throughout the flight with the maximum occurring during the last set of parabolas. However, both males and females return to pre-parabola activity levels after the final parabola. The statistical differences between males and females occur in the vertical group during level flight, both before and after the parabolas, and in the horizontal group for parabolas 11-20.		Figure 4. Activity level for female fruit flies during segments of the KC-135 flight. Error bars indicate standard error. Level flight before and after lasted 9.6 minutes and 24.6 minutes, respectively. Each parabola lasts 20-25 seconds.

**Summary**
The activity level of Drosophila melanogaster (fruit flies) during exposure to microgravity was increased in comparison to activity under Earth’s gravity (1 g). The sounding rocket data indicates an increasing activity level during the few minutes of microgravity, which then levels off or slowly decreases depending upon orientation and sex. The KC-135 data shows an increasing activity level throughout the entire flight. Our results show that orientation (horizontal vs. vertical) had an effect on activity level, particularly during the KC-135 flight, and that sex (male vs. female) had little effect on activity level. However, direct comparisons between the rocket and KC-135 flights are difficult due to the temperature differences. The rocket data was collected at temperatures between 24.5 and 26.0° C while the KC-135 data was collected at 18.0 to 20.0° C. This temperature change probably accounts for the activity level differences observed between the 1 g portions of the rocket and KC-135 data.
Our results indicate that microgravity does increase fruit fly activity and, thus, may account for the previously measured increased aging by male fruit flies exposed to long-term microgravity. However, making extrapolations to the long-term effects of microgravity are difficult since the rocket flight provided slightly less than 4 minutes of microgravity and the KC-135 provided only 20-25 seconds of microgravity accompanied by 30-40 seconds of hypergravity (1.8 g) per parabola. Interestingly, the results from this study are in contrast to previous KC-135 research, using video-taped flies, that indicated a sudden increase in activity upon entering microgravity that subsequently decreased throughout the flight [2].

**Future Work**
Experiments are currently being performed to determine the differences in activity level due to temperature. These results may provide insight into the differences observed during the rocket and KC-135 flights. We are also continuing the data analysis on other KC-135 flights, including video data, which may explain the differences between our results and previous research. Future experiments should examine the effects of long-duration microgravity on fruit fly activity using a GAS can or possibly onboard the ISS. The Canadian Space Agency and NASA are working together to develop a fly laboratory that should fly on the ISS within the next 5-10 years.

**References and Acknowledgments**
References:
1) Benguria, A., Enrique, G., de Juan, E., Ugalde, C., Miquel, J., Garesse, R., and Marco, R. (1996) Microgravity Effects on Drosophila Melanogaster Behavior and Aging. Implications of the IML-2 Experiment. Journal of Biotechnology 47, pp. 191-201; 2) Le Bourg, E., Grimal, A., Fresquet, N., Lints, F.A. (1995) Spontaneous locomotor activity of Drosophila melanogaster at various gravity levels (0 g, 1 g, 1.8 g) during parabolic flights. Behavioural Processes 34, pp. 175-184; 3) Marco, R., Vernos, I., Gonzalez, J., and Calleja, M. (1986) Embryogenesis and Aging of Drosophila Melanogaster Flown in the Space Shuttle, Naturwissenschaften 73, pp. 431-432; 4) Miquel, J. (1985) Effects of microgravity and hypergravity on invertebrate development. NASA Developmental Workshop (K.A. Souza and T.W. Halstead, eds.), Arlington, VA, pp. 7-35, NASA-TM-86756; 5) Miquel, J. and Souza, K.A. (1991) Gravity Effects on Reproduction, Development, and Aging. Advances in Space Biology and Medicine, pp. 71-97, JAI Press Inc

Acknowledgments:
This research is supported by NASA’s Student Launch Program (NASA NGT-5135) and NASA Vermont EPSCoR.

Web Page Design: Mark U. Meissner
University of Vermont: College of Engineering & Mathematics
Last Rev: October 28, 2000
Any comments or suggestions, e-mail: Mark Miller