on Circadian Locomotor Activity in Fruit Flies Reference A.G. Renwick. Acceptable daily intake and regulation of intense sweeteners. 1990.Journal Food Additives And Contaminants. Vol 7, NO. 4, 463-475. Assessing Effects of Mating Status and a White-Eye Mutation, on an Isogenic Background, on Circadian Locomotor Activity in Fruit Flies Sophie Breitbart, Saratoga Springs High School, and Professor Bernard Possidente Skidmore College Biology Department, Saratoga Springs, New York 12866 Discussion Fruit flies (Drosophila melanogaster) are a valuable model system for the analysis of circadian biological clock function. Drosophila circadian clocks in the brain are entrained to the light-dark cycle through multiple photoreceptor pathways (1) , and all these pathways receive light that is filtered through pteridine (red) and ommochrome (brown) eye color pigments. Our research demonstrates that these pigments, removed by the white eye mutation (2) do not play a significant role in determining circadian clock period, or mean levels of activity in either a light-dark cycle or constant darkness. Since our results are based on an isogenic strain comparison, the white eye mutation is not confounded with any other genetic differences between the strains. On the other hand, our results are specific to this particular strain. We also determined, independently of eye color, that mating status does not alter mean activity level or circadian period in males and females, consistent with a previous report for females (3) . This suggests that any endocrinological, neural or behavioral changes that might be induced from mating in Drosophila do not involve circadian clock mechanisms. Driving locomotor behavior. It is probably not necessary, therefore, to isolate virgin flies for analysis in Drosophila Activity Monitors, even though mated females may lay eggs that hatch and release larvae in the apparatus during an experiment. We observed a gender difference in mean activity in constant dark with females displaying more activity than males, but no difference in the 12:12 LD cycle. This is consistent with previous reports of higher activity levels in females, and gender-differences in circadian period that are strain-dependent (3). Finally, we observed no interactions among the white-eye mutation, mating status and gender treatments. We conclude that the frequent use of white-eye mutant strains as controls and background strains for transgenic lines (4) is not likely to bias studies of circadian locomotor behavior. Acknowledgements: We thank Alysia Hildebrandt for identifying and isolating the white-eye mutation in the wild-type isogenic strain used here. Results The white eye mutation had no significant effect on mean activity levels or circadian period. Mating status had no significant effect on mean activity levels or circadian period. Gender had no significant effect on mean activity levels in 12:12 LD or circadian period (tauDD), but mean activity in DD was higher in females (P < 0.05). There were no significant interactions between treatments for any variable. Introduction An isogenic strain of Drosophila melanogaster was to determine how white eye color, gender, and mating status affected circadian locomotor activity. This method tested the white eye mutation against a constant genetic background. Changing the eye pigmentation may have altered the light input to the circadian clock in the brain. We also ask if mating experience changes locomotor activity in males and females. Materials and Methods A spontaneous white eye mutation occurred in an inbred fruit fly strain (fig .1). The mutation complimented a known white eye mutation on the X chromosome. Purebred white and red eyed inbred lines were derived in our lab. Virgin and non virgin red and white-eyed flies of both sexes, aged 1 to 3 days old(n=16 per treatment group, 128 total) were placed in a Drosophila Activity Monitor (fig. 2) on a 12 hours of light and 12 hours of dark cycle (12:12 LD) for 3 days, and for 10 days in constant dark (DD). Activity counts were recorded in a 10 minute bins. The effects of genotype, mating status, and gender on mean activity level in 12:12 LD and DD and free-running circadian period in DD (estimated with Chi-square periodogram) and interactions were analyzed using analysis of variance. Male Virgin Male Non-Virgin n.s. n.s. Fig. 4: Eye color had no significant effect on XLD, XDD, or tauDD in male virgin flies (Left) and male non-virgin flies (Right.) Mating status also had no significant effect, and there were no gender effect on XLD and tauDD. XDD was significantly higher in females (P < 0.05; see Fig. 6 below.) There were no significant interactions between treatments. Female Virgin Female Non-Virgin Fig. 1: Image of a red eyed female (left) and a white eyed male (right.) Fig. 2: Drosophila activity monitor. n.s. n.s. References Ashmore LJ, Sehgal A (2003) A fly’s eye view of circadian entrainment.J. Biol. Rhythms 18:206-216. Lindsley DL, Zimm GG (1992) The genome of Drosophila melanogaster. Academic Press, New York, p. 759. (3) Helfrich-Förster C (2000) Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster – Sex-specific differences suggest a different quality of activity. J Biol Rhythms 15:133-154. (4) Celotto AM, Palladino MJ (2005) Drosophila: a “model” system to study neurodegeneration. Mol Interv. 5:292-303. The Drosophila activity monitors generated an actogram for each fly (fig. 3). Fig. 3: Representative actogram showing three days of activity in 12:12 LD followed by ten days in constant dark (DD.) The Y axis shows consecutive days and the X axis is double-plotted, showing forty eight hours of continuous activity with the second day replotted as the first day on the next line. Fig. 5: Eye color had no significant effect on XLD, XDD, or tauDD in female virgin flies (Left) and female non-virgin flies (Right.) Mating status also had no significant effect, and there were no gender effect on XLD and tauDD. XDD was significantly higher in females (P < 0.05; see Fig. 5 above.) There were no significant interactions between treatments.