山果蝇物种亚群（Drosophila montium species-subgroup）求偶行为及求偶歌进化及其相关基因研究

Premating isolation mainly results from the variation of courtship behavior。Precopulatory courtship is the typical Drosophila courtship that male flies vibrate wing(s) to generate species-specific pulse song before mounting. In our previous study, we found that species in lini clade of montium species-subgroup display only copulatory courtship and produce species-specific sine song after mounting. We further investigated the courtship behavior of kikkawai clade in montium subgroup, species of kikkawai clade showed only copulatory courtship as well. A couple of species in montium subgroup, such as species in auraria and serrate species-complex, have been report to display copulatory courtship. The auraria complex has both precopulatory courtship and copulatory courtship. Based on above phenomena, we propose a hypothesis as that copulatory courtship is dominant in montium subgroup, and sine song plays a crucial role for mate recognition in montium subgroup, these two types of courtship differentiated from montium subgroup. We will test the hypothesis based on the studies on courtship behaviors and courtship songs among 29 species of montium subgroup using 6 species of other subgroups as the outgroup. We will also try to screen copulatory courtship and sine song associated genes using SSH and cDNA microarry. To uncover how the copulatory courtship and precopulatory courtship evolve, and what's role of sine song and pulse song in montium subgroup and in the melanogaster species-group. We hope the results will shed light on understanding of evolution of drosophila courtship behavior and courtship song, and its genetic control.

求偶行为的变异导致生殖前隔离。典型的果蝇预交配求偶precopulatory courtship为雄蝇在乘骑前振翅产生种特异性脉冲歌。我们的研究成果显示，lini进化枝显示独特的交配求偶copulatory courtship并产生雌蝇偏好的种特异正弦歌，kikkawai进化枝也仅在乘骑后产生求偶歌。已知具交配求偶的有auraria和serrate物种复合组的物种，它们均属山果蝇物种亚群，auraria物种复合组兼有两类求偶行为。据此，我们假设：交配求偶在山果蝇物种亚群中普遍存在，乘骑后的正弦歌在配偶识别中起关键作用，两类求偶行为在该物种亚群中分化。本项目拟以山果蝇物种亚群29种，黑腹果蝇物种群6物种亚群代表种为材料，研究其交配行为和求偶歌，以检验以上假设，并阐明这两类求偶行为在该亚群中获得或丢失之进化机制，验证正弦歌与脉冲歌在该亚群求偶中的作用，并筛查与交配求偶行为和正弦歌频率相关基因。

对山果蝇物种亚群42个物种大量样本求偶行为视频和求偶歌录制分析，结果显示该物种亚群存在着三种求偶类型：5个物种为P/C型（P为80%以上雄蝇展现预交配求偶行为，C为100%雄蝇展现交配求偶行为）；24个物种为P-/C型（P-为80%以下雄蝇展现预交配求偶行为）；13个物种为C型。定义了一种新型求偶歌“具结构正弦歌（Structured sine song, S-sine song）”及其参数“单位结构频率（Unit Frequency, UF）”，具有预交配求偶行为物种攀爬前所产生的求偶歌有57.14%为脉冲歌（pulse song），23.81%为S-sine song，19.05%为正弦歌（sine song）；攀爬后所产生的求偶歌40%为具结构正弦歌，40%为普通正弦歌，20%为脉冲歌。这些结果确认了交配求偶行为是山果蝇物种亚群普遍存在的求偶特征，而预交配求偶行为则在该物种亚群中不同程度地丢失；交配求偶中的脉冲歌在该物种亚群有丢失趋势，在歌的结构上，具结构正弦歌居于脉冲歌和正弦歌之间，可能是求偶歌进化中的中间产物。.对P-/C型代表物种D. serrata进行攀爬前后振翅行为和求偶歌分析，第一次求偶攀爬前有27%的雄蝇以小角度振翅产生IPI约31msec的脉冲歌，攀爬后100%雄蝇以大角度振翅产生单位结构频率（UF）约为126/sec的具结构正弦歌；第二次求偶攀爬前100%雄蝇以大角度振翅产生具结构正弦歌。导致雄蝇以大角度振翅产生具结构正弦歌的主要因素为雌蝇的拒绝行为和雄蝇的学习行为。从攀爬前振翅求偶与不振翅求偶雄蝇构建的SSH cDNA文库中选出求偶行为相关候选基因obp56g、14-3-3zeta、Desat1、Argk和Cyp9b2基因进行实时定量PCR（RT-PCR），发现以上除了Argk外，其他基因在攀爬前振翅和不振翅的雄蝇个体中表达量均表现出了显著性差异，为后续的功能验证提供了候选基因。.首次在山果蝇物种亚群中鉴定到有四个物种的雄蝇腹部存在劳伦斯肌肉（Muscle of Lawrence），对具有劳伦斯肌肉的代表物种D. kikkawai进行三代测序，并对控制该类肌肉形成的fruitless（fru）基因进行分析和克隆。.本项目的研究成果有助于解析山果蝇求偶行为和求偶歌的进化规律及遗传机制，具有重要理论意义。