德國蜚蠊生物時鐘掩蓋機制探討(3/3)

由於德國蜚蠊活動行為的表現上，雌 蟲會因為卵巢的發育，而造成活動行為日 週律動被掩蓋的現象，本計劃嘗試深入探 討此掩蓋機制。首先我們進行觸角電位圖 譜 (Electroantennogram, EAG) 的偵測，當 作第二個生物時鐘外在表徵：利用雌蟲之 觸角電位圖譜的偵測，我們可以了解雌蟲 活動行為之掩蓋現象其作用之機制位於生 物時鐘系統的哪一部位。另外因為德國蜚 蠊活動行為的掩蓋現象是生殖行為的生理 活動運作的結果，我們同時選取交尾活性 (mating activity) 作為另一個生物時鐘的外 在表徵來研究掩蓋現象。 其次，德國蜚蠊與雙紋姬蠊這兩個同 胞種在交尾前期的日週律動的表現有很大 的差異，是探討「日週律動掩蓋機制」很 好的材料。在基因層次的研究方面，針對 時鐘基因per 在德國蜚蠊與雙紋姬蠊之 DNA 序列上做比較，希望從時鐘基因per 的分子演化上了解影響生物時鐘表現的因 子為何。 最後，由於掩蓋現象是生物時鐘節律 器 (pacemaker) 的時間訊息在輸出 (output) 的過程中受到生殖因子的影響而 造成活動行為日週律動消失，為了想要知 道生物時鐘與生殖的關係在其它物種的情 況為何，我們比較不同職務的白蟻(生殖型 與非生殖型) 之 PER 、PDF 以及 Corazonin 三種蛋白質在不同時間點的表 現差異(包含日夜變化)，分析其作用位置與 活動行為日週律動訊息傳遞路徑的關係， 進而了解此掩蓋作用之機制，同時藉此研 究對昆蟲之生物時鐘與生殖的關係有更進 一步的了解。The locomotor circadian rhythm was masked by the development of ovaries in female German cockroach (Blattella germanica). This study explores the masking mechanisms of the biological clock in cockroach. Firstly, we proceed the detection of Electroantennogram (EAG) as the second external indicator of circadian clock. We realize the localization of masking mechanism of biological clock based on EAG results. Since the masking phenomenon is result from physiological activity of reproduction, we choose the mating activity as an other indicator to study the masking effect. Secondly, Blattella germanica and B. bisignata are close-related species, but they show huge differences in the expression of circadian rhythm s before mating. The comparative study of the expression of circadian rhythm between two species, is a good theme in the investigation of ecological significance of circadian rhythm. Comparing the DNA sequences of per gene between B. germanica and B. bisignata, we understand which factor influences the 2 expression of circadian clock based on the molecular evolution of per gene. Finally, in order to understand the effect of reproductive factor on the output pathway and masking the circadian rhythm of locomotion, termite are used to investigate the relationship between reproduction and circadian clock in non-cockroach species. We compare the expression of three clock gene (per, pdf and corazonin) in individual of different casts at different time point. This result shows the locations where these clock genes the interaction takes place between the expression and transmission pathway of time signal for locomotion. Based on the above research, the masking mechanism are understood clearly

蛻皮激素對德國蜚蠊與雙紋姬蠊之活動行為日週律動的影響(2/3)

德國蜚蠊雌成蟲在活動行為的表 現上，會因卵巢的發育而出現活動行為 日週律動被掩蓋的現象。由於德國蜚蠊 卵巢的掩蓋效應是由荷爾蒙系統所調 控，推測卵巢所分泌最大量的激素「蛻 皮激素」就是掩蓋因子。為了了解蛻皮 激素如何影響活動行為，必須先分析蛻 皮激素之受器基因，因為蛻皮激素會調 控特定組織的蛻皮激素受器，進而影響 下游基因的表現。本年度的工作是進行 蛻皮激素之受器 (ecdysone receptor, EcR) 的分析，利用源自於鱗翅目的抗 體進行 western bloting；接著以免疫染 色的方法找出蛻皮激素在德國蜚蠊成蟲 體內作用之位置；最後做蛻皮激素受器的 蛋白質定序。前述實驗流程在雙紋姬蠊 也進行相同的操作。由於德國蜚蠊與雙 紋姬蠊處女雌蟲的日週律動表現上出 現差異，比較這兩種蜚蠊之蛻皮激素與 掩蓋效應的關係，可以了解造成日週律 動表現在種間差異的生理機制為何。The expression of locomotor circadian rhythm in female German cockroach is masked by the development of ovaries. Since the masking effect is mediated through the regulation of hormone, we propose the ecdysone, the major hormone secreted from ovaries, is the masking factor. Firstly, by analyzing the ecdysone receptor (EcR) gene to realize ecdysone how to influence the locomotor circadian rhythm, because ecdysone will control the EcR gene which influence expression of the gene on the downriver. Firstly, using the antibody from Lepidoptera to work the western bloting. In addition, we will employ immunostain method against ecdysone receptor to reveal the action sites. These sites may shine light on the interference of circadian signals with masking factor-ecdysone. In order to prove the masking role of ecdysone, ovariectomized females who showed unmasked locomotor circadian rhythm will be treated with ecdysone analog to regain the masked rhythm. Finally, protein sequencing of EcR was done. Since the sibling species of the Germany cockroach, the double-striped cockroach is a feral species and expresses locomotor circadian rhythm in virgin females, it is a great advantage to use this species as a comparative study to further demonstrate the function of ecdysone on the locomotor circadian rhythm. The same experimental procedures, therefore, will be repeated on the double-striped cockroach. The main focus of this proposal is to investigate the physiological mechanism of masking effect on the locomotor circadian rhythm by ecdysone