1. Genetic Role in Geotactic Behavior via Pigment Dispersion Factor (pdf) Pathway in Fruit Flies (Drosophila melanogaster) Laura Boon and Tatiana Soboleva (Department of Biological Sciences) Adam Voss, Graduate Mentor (Department of Biological Sciences) Daniel P. Toma, Faculty Mentor (Department of Biological Sciences) Abstract In the early 1960’s, Dr. Jerry Hirsch demonstrated a genetic basis for behavior using fruit fly lines he selected for an extreme response to gravity (geotaxis). Geotactically positive fruit flies tend to travel in the direction of gravity (Lo), and geotactically negative individuals will travel against gravity (Hi) through a vertical (T-choice) maze. These established Hi and Lo flies have since been used as models for the genetic basis of complex behavior. Toma et al., 2002 found a significant effect of the Pdf protein in the geotaxis behavioral response. Flies unable to make Pdf protein are geotactically high and tend to travel against gravity. We have identified several proteins within the literature that interact with Pdf and have obtained mutant fly lines that are either unable to make or make for than normal amounts of these proteins. These are each being bred into a geotactically neutral laboratory line (Canton-S, CS) to test the effect of these individual proteins on complex behavior. We have successfully bred and run a control group (CS) and 10 different mutant fly lines with only five mutations left to finish breeding and collect maze run data on. Any deviation from CS scores in the maze by these mutant flies indicates a possible relationship between the particular proteins affecting geotaxis behavior. We noticed significant patterns in five fly lines so far, which include two timeless mutant lines (Tim4- 1A and Tim7-5B), Nep1, vrille, and robo. However, final statistic analysis has not been complete for all mutant runners. Methodology Set up breeding scheme in order to normalize mutant line onto a geotactically neutral control background which is consistent for each mutation Cross flies carrying experimental mutation with flies expressing known and visibly traceable mutations Select offspring known to carry mutation of interest as well as traceable marker Collect normalized flies known to carry only the mutation of interest Run selected flies through geotaxis maze and collect data Statistically analyze data on behavioral response to gravity Figure1: Figure shows geotaxis maze with one starting point and 15 exits. The selected flies are tested in runs of 110 flies alternating males and females. 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 High Neutral Low Start Light source Figure 2Figure 3 Figure 4 Figure 5 Conclusion Lark 01 gene mutation in female fruit flies presents on average no difference in geotactic behavior compared to a slightly positive geotactic response of male individuals, the data being normalized to the CS mutants. Nep2 gene mutation shows on average a positive geotactic response of both female and male fruit flies, data being normalized to CS mutants. Female individuals are showing a more prominent negative geotactic response compared to the males of the same gene mutation. Tim7-5B and Tim4-1A gene mutations show a negative geotactic behavior in both male and female fruit flies, data normalized against CS mutants. Males showing a more noticeable positive geotactic response. Figure 2 indicated that Pdf mutation further confirms the results form Toma et al. 2002. The collected data on Nep4, Vri, Nep1, robo, cyc, and Dh31 mutations has yet to be statistically analyzed and reported. Discussion The analyzed genetic mutations Nep2, Tim7-5B, and Tim4-1A show impact on geotactic behavior when compared to the CS average, statistical analysis is yet to be carried out on the collected data. The data is yet to be collected and examined on Clock ar, tim 01, Pdp 1, Nep5, and Nep3 mutants. In case the results stated do show statistically different results from CS, other genes related to Nep2 and Tim can be used for further geotactic analysis. Trial AveragesPercentages Tube #MalesFemalesMalesFemales 1527.623.428.63%23.64% 1418.823.219.50%23.43% 1310.812.811.20%12.93% 12810.28.30%10.30% 1168.26.22%8.28% 104.664.77%6.06% 94.244.36%4.04% 82.83.82.90%3.84% 72.42.62.49%2.63% 63.823.94%2.02% 51.20.81.24%0.81% 42.20.62.28%0.61% 31.60.61.66%0.61% 20.80.40.83%0.40% 11.60.41.66%0.40% Totals96.499100.00% AveragesPercentages Tube #MalesFemalesMalesFemales 1514.81915.13%20.26% 147.6167.77%17.06% 1312.213.212.47%14.07% 1211.210.211.45%10.87% 119.26.49.41%6.82% 108.47.68.59%8.10% 963.86.13%4.05% 85.83.25.93%3.41% 74.63.84.70%4.05% 6545.11%4.26% 54.824.91%2.13% 432.23.07%2.35% 32.41.62.45%1.71% 21.80.41.84%0.43% 110.41.02%0.43% Totals97.893.8100.00% AveragesPercentages Tube #MalesFemalesMalesFemales 151311.813.83%11.78% 149.411.610.00%11.58% 1369.86.38%9.78% 127.287.66%7.98% 115.87.86.17%7.78% 10912.49.57%12.38% 98.66.29.15%6.19% 86.24.86.60%4.79% 75.64.85.96%4.79% 65.87.86.17%7.78% 544.24.26%4.19% 44.44.64.68%4.59% 33.22.23.40%2.20% 2222.13%2.00% 13.82.24.04%2.20% Totals94100.2100.00% AveragesPercentages Tube #MalesFemalesMalesFemales 1536.438.639.82%40.38% 1414.219.215.54%20.08% 137.210.47.88%10.88% 125.25.85.69%6.07% 116.84.27.44%4.39% 105.22.45.69%2.51% 93.834.16%3.14% 82.422.63%2.09% 71.831.97%3.14% 62.42.62.63%2.72% 51.62.21.75%2.30% 411.21.09%1.26% 3101.09%0.00% 20.80.20.88%0.21% 11.60.81.75%0.84% Totals91.495.6100.00% Figure 2 The Pdf chart shows the difference between scores calculated for mutant fly runs compared to CS normal (control) fly line. The Pdf mutation data table displays average placement of individuals for all five trials performed for males and females independently of each other. Our data showed similar results to the original data collected by Toma et al which further confirmed their previous findings. Resources 1.) Toma, White, Hirsch, Greenspan. “Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait.” Nature Genetics Vol31. (August 2002) : p.349-53. Scholarly article. 2.) Klug, Cummings, Spencer, Palladino. “Concepts of Genetics.” 9th Ed. San Fransisco: Pearson Benjamin Cummings, (2009). Textbook. 3.) Park, Jae H, Hall, Jeffrey C. “Isolation and Chronobiological Analysis of a Neuropeptide Pigment – Disbursing Factor Gene in Drosophila Melanogaster.” J. Biol Rhythms Vol13.No3 (1998) : p.219-28. Scholarly article. 4.) Bahn, Jae Hoon, Lee Gyunghee, Park, Jae H. “Comparative Analysis of PDF-mediated Circadian Behaviors Between Drosophila melanogaster and D. virilis.” Genetics Vol181.No3 (March 2009) : p.965-75. Scholarly article. 5.) Weizmann Institute of Science. http://genecards.org, Department of Molecular Genetics. Web. (1996-2010). 6.) NNF Center for Protein Research. http://string-db.org, STRING 9.0 (Search Tool for the Retrieval of Interacting Genes/Proteins). Web. (2011). 7.) National Center for Biotechnology Information. http://ncbi.nlm.nih.gov, Bioinformatics. National Library of Medicine. Web. (2009). Acknowledgements We would like to say a very special thank you to Adam Voss, our graduate mentor, for all of his help ensuring that we had a successful project. His guidance, support and leadership have made it possible for us to finish a two-year experimental process. We also thank Dr. Toma, our faculty mentor, for allowing us to continue his groundbreaking research. We have learned lessons from both of our mentors that will be carried far beyond this project. We would like to thank the University of Minnesota, Mankato and the Undergraduate Research Committee for allowing us to carry out this experiment and allowing us to present our findings to our peers. This document is available in alternative format to individuals with disabilities by calling Accessibility Resources at 507-389-2825 (V), 800-627-3529 or 711 (MRS/TTY). Figure 3 The Lark 01 chart shows the difference between scores calculated for mutant fly runs compared to CS normal (control) fly line, and the Lark 01 mutation data table displays average placement of individuals for all five trials performed for males and females independently of each other. Our data and trial runs showed no significant change in geotactic behavior when this mutation was induced. Figure 4 The Nep 2 chart displays differences between scores calculated for mutant fly runs compared to CS normal (control) fly line. The Nep 2 mutation data table displays average placement of individuals for all five trials performed for males and females independently of each other. Our data and trial runs reflect a positive geotactic response when flies were induced with the Nep 2 mutation. This indicates their behavioral response was affected by this mutation. Figure 5 The Tim7-5B chart shows calculated differences between scores for mutant fly runs compared to CS normal (control) fly line. The Tim7-5B mutation data table displays average placement of individuals for all five trials performed for males and females independently of each other. Data and trial runs revealed a negative geotactic response when mutant flies were observed in the maze. This indicates a behavioral change occurred when the mutation was introduced.