科學家已研究果蠅的各種行為數十年，但是產生行為的神經迴路仍未被完整地了解。透過光遺傳學的方法，利用自動雷射追蹤及光遺傳調控系統(ALTOMS)，使我們可以在短時間內篩選出與特定行為相關的神經元。經由ALTOMS激發特定被挑選的GAL4啟動子標定的神經，我們發現果蠅會分別做出公母特有的生殖行為，例如：公果蠅求偶行為中的振翅、交配動作，或是母果蠅將產卵器伸出並將腹部往下彎的產卵動作。為了確認上述的神經與交配或產卵行為相關，我們以遺傳學的技術去抑制這些神經後觀察果蠅行為是否變化，實驗結果顯示交配率及下蛋數量皆明顯下降。另一方面，我們也想知道上述神經是否與fruitless有關，出乎意料的是，儘管下蛋與求偶行為屬於雌性及雄性分別專屬的動作，結果卻與調控果蠅雌雄相異性的轉錄因子fruitless無關，證明這些神經並不隸屬fruitless的神經迴路。最後，我們以共軛焦顯微鏡觀察神經在果蠅腹部神經管束以及大腦的分布，並利用不同GAL80 lines去找出與求偶及下蛋行為相關的神經傳導物。未來我們可以運用這些GAL4 line果蠅進一步研究求偶與下蛋行為的神經迴路，讓我們更深度地了解果蠅神經網路連結體的功能。

Diverse behaviors in Drosophila Melanogaster have been studied for many years, the neural circuits behind these behaviors, however, are still not fully understood. New developed automated laser tracking and optogenetic manipulation system (ALTOMS) provides us a platform to high-throughput screen multiple lines with different behaviors. By activating neurons labeled by different GAL4 driver lines, we verified a couple of lines with specific reproductive behaviors, including abdomen bending and wing extension in males and ovipositor extension in females, implying their functions in courtship and egg laying behaviors respectively. We further modulated the activities of target neurons by expressing temperature-sensitive shibire gene, and the results indicated that thermogenetic inhibition of these specific neurons decreased the copulation rate as well as the number of laying eggs in corresponding lines. Surprisingly, while both behaviors are sexually dimorphic, the intersection study suggested that none of these neurons is fruitless positive. The neural anatomies of these targeting GAL4 lines were also examined under confocal microscopy. Finally, in order to narrow down the target neurons, the molecular identities of the labeled neurons were characterized by multiple GAL80 driver lines for different neurotransmitters. Identifying these candidate GAL4 lines led us to reveal new circuits involved in courtship and egg laying behaviors, which would eventually enhance our understanding of functional connectome in fruit fly.

1. Manoli, D.S., Meissner, G.W., and Baker, B.S. (2006). “Blueprints for behavior: genetic specification of neural circuitry for innate behaviors.” Trends in Neurosciences 29, 444-451

2. Potdar, S. and Sheeba, V. (2013). “Lessons from Sleeping Flies: Insights from Drosophila melanogaster on the Neuronal Circuitry and Importance of Sleep.”
Journal of Neurogenetics 27, 23-42 |

3. Brand, A.H. and Perrimon, N. (2003). “Targeted gene expression as a means of altering cell fates and generating dominant phenotypes” Development 118, 401-415;

4. Hoang, T.T., Karkhoff-Schweizer, R.R., Kutchma, A.H. and Schweizer, H.P. (1998). “A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants.” Gene 212, 77-86

5. Clark, D.L. and Morjan, C.L. (2001). “Attracting female attention: the evolution of dimorphic courtship displays in the jumping spider Maevia inclemens (Araneae: Salticidae).” The Royal Society 268, 1484

6. Muller, M. N. and Wrangham, R.W. (2009). “Sexual Coercion in Primates and Humans.” Harvard University Press

7. Scholes, E. (2008). III “Structure and composition of the courtship phenotype in the bird of paradise Parotia lawesii (Aves: Paradisaeidae).” Zoology 111, 260-278

8. Bastock, M. and Manning, A. (1955). “The Courtship of Drosophila melanogaster.” Behaviour 8, 85-111

9. Von Schilcher, F. (1976). “The behavior of cacophony, a courtship song mutant in Drosophila melanogaster.” Behav Biol. 17, 187-96.

10. Spieth, H.T. (1952). “Mating behavior within the genus Drosophila (Diptera).” Bulletin of the AMNH 99, article 7

11. Economos, A.C., Miquel, J., Binnard, R. and Kessler, S. (1979). “Quantitative analysis of mating behavior in aging male drosophila melanogaster.” Mechanisms of Ageing and Development10, 233-240

12. Amrein, H. (2004). “Pheromone perception and behavior in Drosophila.” Current Opinion in Neurobiology 14, 435-442

13. Hall, J. C. (1986). “Learning and rhythms in courting, mutant Drosophila.” Trends in Neurosciences 9, 414-418

14. Kulkarni, S.J., Steinlauf, A.F. and Hall, J.C. (1988). “The dissonance mutant of courtship song in Drosophila melanogaster: isolation, behavior and cytogenetics.” Genetics 118 267-285

15. Kim, W.J., Jan, L.Y. and Jan, Y.N. (2013). “A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating.” Neuron 5, 1190-1205

16. Shankar, S., Chua, J.Y., Tan, K.J., Calvert, M.E., Weng, R., Ng, W.C., Mori, K. and Yew, J.Y. (2015). “The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit.” eLife 4, e06914

17. Zhang, S.X., Rogulja, D. and Crickmore, M.A. (2016). “Dopaminergic Circuitry Underlying Mating Drive.” Neuron 91 168-181

18. Burtis, K.C. (1993). “The regulation of sex determination and sexually dimorphic differentiation in Drosophila.” Current Opinion in Cell Biology 5 1006-1014

19. Ferveur, J.F. (1995). “Genetic feminization of brain structures and changed sexual orientation in male Drosophila.” Science 267, 902-905

20. Waterbury, J.A., Jackson, L.L. and Schedl, P. (1995). “Analysis of the Doublesex Female Protein in Drosophila melanogaster: Role in Sexual Differentiation and Behavior and Dependence on Intersex.” Genetics 152, 1653-1667

21. Ryner, L.C. (1996). “Control of Male Sexual Behavior and Sexual Orientation in Drosophila by the fruitless Gene.” Cell 87, 1079-1089

22. Kimura, K.I., Ote, M., Tazawa, T. and Yamamoto, D. (2005) “Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain.” Nature 438, 229-233

23. Demir, E. and Dickson, B.J. (2005). “fruitless Splicing Specifies Male Courtship Behavior in Drosophila.” Cell 121, 785-794

24. Bray, S and Amrein, H. (2003). “A Putative Drosophila Pheromone Receptor Expressed in Male-Specific Taste Neurons Is Required for Efficient Courtship.” Neuron 39, 1019-1029

25. Lin, H., Mann, K.J., Starostina, E., Kinser,R.D. and Pikielny , C.W.(2005).” A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones.” Proc. Natl. Acad. Sci. 102 (36) 12831-12836

26. Miyamoto, T. and Amrein, H. (2008). “Suppression of male courtship by a Drosophila pheromone receptor.” Nature Neuroscience 11, 874–876

27. Koganezawa, M., Haba, D., Matsuo, T. and Yamamoto, D. (2010). “The Shaping of Male Courtship Posture by Lateralized Gustatory Inputs to Male-Specific Interneurons.” Current Biology 20,1-8

28. Thistle, R., Cameron, P., Ghorayshi, A., Dennison, L. and Scott, K. (2012). “Contact Chemoreceptors Mediate Male-Male Repulsion and Male-Female Attraction during Drosophila Courtship.” Cell 149, 1140-1151

29. Vijayan, V., Thistle,R., Liu , T., Starostina , S. and Pikielny , C.W (2014). “Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity.” PLoS Genetics10(3)

30. Yu, J.Y., Kanai, M.I., Demir, E., Jefferis, G.S., and Dickson, B.J. (2010). “Cellular Organization of the Neural Circuit that Drives Drosophila Courtship Behavior.” Current Biology 20, 1602-1614

31. Clowney, E.J., Iguchi, S., Bussell, J.J., Scheer, E. and Ruta, V. (2015). “Multimodal Chemosensory Circuits Controlling Male Courtship in Drosophila.” Neuron 87, 1036-1049

32. Siwicki, K.K. and Kravitz, E.A. (2008). “Fruitless, doublesex and the genetics of social behavior in Drosophila melanogaster” Current Opinion in Neurobiology
19, 200-206

33. Manoli, D.S., Foss, M., Villella, A., Taylor, B.J., Hall, J.C. and Baker, B.S. (2005) “Male-specific fruitless specifies the neural substrates of Drosophila courtship behavior.” Nature 436, 395–400

34. Kohatsu, S., Koganezawa, M. And Yamamoto, D. (2011). “Female Contact Activates Male-Specific Interneurons that Trigger Stereotypic Courtship Behavior in Drosophila.” Neuron 69, 498-508

35. Philipsborn, A.C., Liu,T, Yu, J.Y., Masser,C., Bidaye, S.S. and Dickson, B.J. (2011). “Neuronal Control of Drosophila Courtship Song.” Neuron 69, 509-522

36. Clyne, J.D. and Miesenböck, G. (2008). “Sex-Specific Control and Tuning of the Pattern Generator for Courtship Song in Drosophila.” Cell 133, 354-363

37. Chen, P.S., Stumm-Zollinger, E., Aigaki, T., Balme, J., Bienz, M. and Böhlen, P. (1988). “A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster.” Cell 54, 291-298

38. Connolly,K. and Cook,R. (1973). “Rejection Responses by Female Drosophila melanogaster: Their Ontogeny, Causality and Effects upon the Behaviour of the Courting Male.” Behaviour 44, 142-166

39. Herndon, L.A. and Wolfner, M.F. (1995). “A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating.” Proc. Natl. Acad. Sci. 92 (22) 10114-10118

40. Ribeiro, C. and Dickson, B.J. (2010). “Sex Peptide Receptor and Neuronal TOR/S6K Signaling Modulate Nutrient Balancing in Drosophila.” Current Biology 20, 1000-1005

41. Joseph, R.M., Devineni, A.V., King I.F. and Heberlein, U. (2009). “Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.” Proc. Natl. Acad. Sci.106 (27) 11352-11357

42. Dweck, K.M., Ebrahim,S.M., Kromann,S., Bown, D., Hillbur,Y., Sachse,S., Hansson, B.S. and Stensmyr, M.S. (2013). “Olfactory Preference for Egg Laying on Citrus Substrates in Drosophila.” Current Biology 23, 2472-2480

43. Yang, C.H., Belawat, P., Hafen, E., Jan, L.Y. and Jan, Y.N. (2008). “Drosophila Egg-Laying Site Selection as a System to Study Simple Decision-Making Processes.” Science 319, 1679-1683

44. Stensmyr, M.C., Dweck, K.M., Farhan, A., Ibba, I., Strutz, A., Mukunda, L., Linz, J., Grabe, V., Steck, K., Lavista-Llanos, S., Wicher, D., Sachse, S., Knaden, M., Becher, P.G., Seki, Y. and Hansson, B.S. (2012). “A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila.” Cell 151, 1345-1357

45. Rideout, E.J., Dornan, A.J., Neville, M.C., Eadie, S. and Goodwin, S.F. (2010). “Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster.” Nature Neuroscience 13, 458–466

46. Lewis, E.B. and Bacher, F. (1968). “Method of feeding ethyl methane-sulphonate to Drosophila males.” Dros. Inf. Serv. 43, 193-194

47. Neely, G.G., Hess, A., Costigan, M., Keene, A.C. and Goulas, S. (2010). “A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionary-conserved pain gene.” Cell 143(4): 628–638.

48. Flood, T.F., Gorczyca, M., White, B.H., Ito, K. and Yoshihara, M. (2013). “A Large-Scale Behavioral Screen to Identify Neurons Controlling Motor Programs in the Drosophila Brain.” G3 3(10): 1629–1637.

49. Hoopfer, E.D., Jung, Y., Inagaki, H.K., Rubin, G.M. and Anderson, D.J. (2015). “P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila.” eLife 4: e11346

50. Wu, M.C., Chu L.A., Hsiao, P.Y., Lin, Y.Y., Chi, C.C., Liu, T.H., Fu, C.C. and Chiang, A.S. (2014). “Optogenetic control of selective neural activity in multiple freely moving Drosophila adults.” Proc. Natl. Acad. Sci. 111 (14) 5367-5372

51. Sadaf, S., Reddy, O.V., Sane, S.P. and Hasan, G. (2015). “Neural Control of Wing Coordination in Flies.” Current Biology 25, 80-86

52. Dow, M. A. and Schilcher, F. (1975). “Aggression and mating success in Drosophila melanogaster.” Nature 254, 511–512

53. Finley, K.D., Taylor, B.J., Milstein, M. and McKeown, M. (1997). “dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster.” Proc. Natl. Acad. Sci. 94 (3) 913-918

54. Yapici, N., Kim, Y.J., Ribeiro, C. and Dickson, B.J. (2008). “A receptor that mediates the post-mating switch in Drosophila reproductive behavior.” Nature 451, 33–37

55. Ram, K.R. and Wolfne, M.F. (2007). “Sustained Post-Mating Response in Drosophila melanogaster Requires Multiple Seminal Fluid Proteins.” PLoS Genetics 3(12) 2428-2438

56. Rezával, C., Pavlou, H.J., Dornan, A.J., Chan, Y.B., Kravitz, E.A. and Goodwin, S.F. (2012). “Neural Circuitry Underlying Drosophila Female Postmating Behavioral Responses.” Current Biology 22, 1155-1165

57. Haussmann, I.U., Hemani, Y., Wijesekera, T., Dauwalder, B. and Soller, M. (2013). “Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours.” Proc. R. Soc. B 280