Potthoff

Patterns of locomotion activity during hypergravity in larval Drosophila melanogaster.

Potthoff, Matthew J., and James N. Thompson, jr. Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019.

Drosophila is a useful model system for studying the biological basis of locomotion and factors that influence it (e.g., Sokolowski, 2001). Our interest in locomotion is through the role it may play in adaptation to long-term culture in the special conditions of a Space Station environment. One of those special conditions is the periodic exposure to hypergravity as a consequence of shuttle launches and other actions. Understanding how Drosophila responds to hypergravity might also lead to predictions about activity in microgravity. Adult Drosophila are highly mobile and can detect and respond to changes in gravity (LeBourg et al., 1999). Although larvae are slower and more limited in movement than adults, we hypothesize that hypergravity can still be a stress that will impede their mobility significantly.

In this study, larval locomotion was observed and videotaped while the flies were being spun in a centrifuge at 4 g. Larval locomotion rate was measured by digitizing the larval position coordinates continuously during experimental periods of 70 minutes each (10 min taped acclimation; 30 min at 4 g; 30 min at 1 g). We found that locomotion rate declined during exposure to 4 g, and larvae were frequently observed to exhibit an enhanced escape-like behavior (positive geotaxis) during and after exposure to hypergravity.

A 22"-diameter centrifuge was constructed with a metal armature attached to a bearing assembly (Figure 1). A pulley system was used to reduce the rate of revolution. The drive wheel was attached to the power source, a variable-speed drill regulated by a rheostat. RPM was quantified with a photocontact tachometer, and a gravitational force of 4 g could be maintained for extended periods. Larval D. melanogaster were placed in a plastic arena where their behavior could be observed and videotaped using a miniature high resolution videocamera (ALM-2453 2.4 GHz Wireless Miniature Camera, 10´ mag; Figure 2).

Canton-S 3^rd instar larvae were collected from stocks that had been set up 7 days in advance. Canton-S stocks were chosen because they are believed to have no associated behavioral mutations. Experiments were performed at approximately the same time each day (between 12:00 and 1:00 p.m.) to avoid variation in circadian rhythm. Larvae were placed on a glucose medium inside of a 2.5 cm diameter circular arena. The arena was covered with a piece of non-reflective glass and clamped down to prevent the escape of larvae. The larval container was attached to the metal armature of the centrifuge where the larvae were allowed to acclimate for 15 minutes at approximately 25°C. After acclimation, larvae were videotaped for 10 minutes as a control. The centrifuge was then turned on and allowed to reach a force of 4 g for 30 minutes (treatment) while being videotaped. The centrifuge was then turned off and the larvae remained videotaped for an additional 30 minutes (post-treatment).

Figure 1. Centrifuge for videotaping larval behavior while maintaining low levels of gravitational stress. 1 – Phototachometer; 2 – Rheostat; 3 – Variable-speed drill; 4 – Pulley system; 5 – Drive wheel; 6 – Camera power source; 7 – Metal armature; 8 – Magnification system; 9 – Larval container; 10 – Cool sticker.

Figure 2. Magnification system. 1 – Camera; 2 – Magnification lenses (5´, 10´ or 15´); 3 – Larval arena.

The videotape was placed in a S-VHS Panasonic AG-7750 VCR and movements were recorded by hand using a transparency over a monitor to plot the points of travel. Data points were numbered sequentially at each point they turned and were labeled to identify when five minutes had been completed under each regimen (control, treatment, and post-treatment). The transparencies were scanned into the computer and data points were digitized (Figure 3) using a morphometrics program maintained by F. James Rohlf at SUNY Stony Brook (http://life.bio.sunysb.edu/morph/; tpsDIG for data acquisition; tpsUtil for utility programs). Analysis of the data was performed using Morpheus et al. program (Slice, 2000), which used a designed syntax that measured the distances traveled between points (Figure 4). These data were then exported to an Excel file, where the accumulated distance traveled for each 5-minute interval was recorded.

This larval behavioral study exhibited a range of activity levels. Since both an increase in activity and a lack thereof could be significant, calculations were made with periods of activity and also separately with the exclusion of larvae that exhibited no movement during some of its 5-minute intervals. Furthermore, since the levels of locomotion could fluctuate or show trends within the treatment and post-treatment time intervals, each 30-minute section was broken down further into 6 five-minute sections. Tables 1 and 2 compare the larval locomotion rates between selected periods before, during, and after exposure to 4 g hypergravity treatment, where Table 1 includes periods of no movement and Table 2 excludes these periods.

Several conclusions can be made from the results of the larval locomotion when considering the whole data set. First, larval locomotion rate declined during exposure to 4 g treatment (Table 1). Second, the first 5 minutes after treatment (post-treatment) were significantly reduced in locomotion (last 5 minutes of treatment vs first 5 minutes of post-treatment (with and without zeros). Third, the first 5 minutes of the post-treatment were significantly reduced from the second 5 minutes of the post-treatment (with zeros)(P = 0.042, t = 2.1). Finally, the control was significantly different from the post-treatment as a whole (with zeros)(P = 0.012, t = 2.5), but the last 10 minutes of the post-treatment was not significantly different from the control (P = 0.244, t = 1.2).


Figure 3. Marked points of larval movement.	Figure 4. Digitized sequence of movement.

Table 1. Comparison of larval locomotion rates at selected periods before, during, and after exposure to 4 g hypergravity treatment. Periods of no movement are included.

Comparison
	Mean ± s.d. (n)		Mean ± s.d. (n)	t	df	P

Control	1993 ± 1280 (62)	At 4g	1727 ± 1516 (186)	1.2	246	0.216
At 4 g	1727 ± 1516 (186)	Post 4g	1476 ± 1428 (186)	1.6	370	0.101
Control	1993 ± 1280 (62)	Post 4g	1476 ± 1428 (186)	2.5	246	0.012
At 4g (Final 5 min)	1628 ± 1385 (31)	Post 4g (First 5 min)	845 ± 844 (31)	2.7	60	0.009
Control	1993 ± 1280 (62)	Post 4g (Last 10 min)	1694 ± 1534 (62)	1.2	122	0.244
Post 4g (First 5 min)	845 ± 844 (31)	Post 4g (Second 5 min)	1395 ± 1204 (31)	2.1	60	0.042

Table 2. Comparison of larval locomotion rates at selected periods before, during, and after exposure to 4 g hypergravity treatment. Periods of no movement are not included.

Comparison
	Mean ± s.d. (n)		Mean ± s.d. (n)	t	df	P

Control	2200 ± 1240 (48)	At 4g	1833 ± 1479 (144)	1.5	190	0.123
At 4 g	1833 ± 1479 (144)	Post 4g	1832 ± 1420 (144)	0.01	286	0.994
Control	2200 ± 1240 (48)	Post 4g	1832 ± 1420 (144)	1.6	190	0.111
At 4g (Final 5 min)	1834 ± 1378 (24)	Post 4g (First 5 min)	1025 ± 875 (24)	2.4	46	0.019
Control	2200 ± 1240 (48)	Post 4g (Last 10 min)	2143 ± 1471 (48)	0.2	94	0.84
Post 4g (First 5 min)	1025 ± 875 (24)	Post 4g (Second 5 min)	1618 ± 1248 (24)	1.9	46	0.06

We have a hypothesis to explain this behavior. The last 10 minutes of the post-treatment were not significantly different from the control, but the first 5 minutes deviated significantly from the control and the first 5 minutes also demonstrated a reduction in locomotion rate. This suggests that the 4 g treatment stimulates an immediate reduction in locomotion, followed by a gradual recovery (recovery time and movement may vary between larvae). During the 4 g treatment, the larvae may wander slowly in order to attempt to escape the stress.

When the centrifuge is turned off (first 5 min of post-treatment), the larva reduces movement because now it is located where the stress can no longer be sensed. After a short time period, the larvae gradually increase wandering movement and do not sense the stress anywhere, thereby returning wandering movement to a normal rate (last 10 minutes of post-treatment). Another possible explanation of these results is the escape-like behavior of the larvae following 4 g treatment, in which some larvae attempted a positive geotactic behavior, which reduced their wandering movement; however, these behaviors were not frequent and they were not quantified in this study.

Table 3. Average locomotion rates (mean ± s.d.) for 5 minute intervals. Rates are given for all data (n = 31), as well as for only those in which movement was occurring (i.e., excluding zeros; (n = 24).

Time Period	All Data	Excluding Zeros

Control
1^st 5 min	1866 ± 1183	2069 ± 1155
2^nd 5 min	2119 ± 1377	2330 ± 1331

4 g Treatment
1^st 5 min	2226 ± 1962	2243 ± 1842
2^nd 5 min	1734 ± 1697	1743 ± 1578
3^rd 5 min	1523 ± 1117	1567 ± 1203
4^th 5 min	1469 ± 1323	1615 ± 1383
5^th 5 min	1779 ± 1463	1995 ± 1454
6^th 5 min	1628 ± 1385	1834 ± 1377

Post-treatment
1^st 5 min	845 ± 844	1025 ± 875
2^nd 5 min	1395 ± 1203	1618 ± 1247
3^rd 5 min	1544 ± 1530	1913 ± 1550
4^th 5 min	1685 ± 1602	2150 ± 1532
5^th 5 min	1756 ± 1598	2218 ± 1519
6^th 5 min	1633 ± 1511	2067 ± 1449

In order to verify these results, more larvae will be treated under higher g levels with increased time of exposure to treatment, and the post-treatment time of study will be lengthened to examine this recovery effect in more detail. Treatment at 4 g is stressful enough to stimulate escape reactions, and we predict that increased hypergravity and exposure time will make these even stronger.

Acknowledgments: We thank Wendal Porter (OU) for his specialties in engineering and construction; Barbara Safiejko-Mroczka (OU) for letting us use her equipment to trace larval movement; Nicholas Mascie-Taylor (University of Cambridge) for his expertise in data interpretation; Max Sanchez from Ames Research Center for his useful discussions; and the University of Oklahoma Zoology Department for their facilities and materials. American Society for Gravitational and Space Biology provided a travel grant to MJP to present the initial results, and an Undergraduate Research Opportunities Program (UROP) grant supported construction of the centrifuge. Funded by NASA grant NAG 2-1427.

References: LeBourg, E., and N. Minois 1999, Exper. Gerontol. 34: 157-172; Rohlf, James F. Morphometrics at SUNY Stony Brook. http://life.bio.sunsb.edu/morph/ ; Slice, D.E., 2000, Morpheus et al.: Software for Morphometric Research. Revision 01-30-98. Department of Ecology and Evolution, State University of New York, Stony Brook, New York; Sokolowski, M.B., 2001, Nature Rev. Genet. 2: 879-890.

	Potthoff, Matthew J., and James N. Thompson, jr. 2002. Patterns of locomotion activity during hypergravity in larval Drosophila melanogaster. Dros. Inf. Serv. 85: 84-87.
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Time Period

All Data

Excluding Zeros

Control

Post-treatment