德國蜚蠊昇海藻糖激素於生殖與抗氧化逆境反應之角色

Abstract

昇海藻糖激素是一種胜肽荷爾蒙，屬於降脂激素家族，其主要功能是促使昆蟲體內海藻糖的生成。本論文研究是在探討德國蜚蠊昇海藻糖激素於生殖與抗氧化逆境反應的功能。該研究分別選殖出昇海藻糖激素與其受器基因，接著利用核醣核酸干擾技術來靜默基因表現以研究其功能。本研究首先證實昇海藻糖激素調控昆蟲體內海藻糖的恆定性，當昇海藻糖激素基因或其受器基因受到核糖核酸干擾而抑制後，德國蜚蠊血淋巴中海藻糖濃度顯著地降低。此外，處女雌蟲血淋巴海藻糖濃度在每個生殖週期有逐漸上升的趨勢，而且與卵巢發育具有正相關性；經實驗驗證，昇海藻糖激素具有調節生殖週期內海藻糖濃度上升的腳色。昇海藻糖激素也會影響雌蟲排出卵鞘的行為，因為處女雌蟲體內的昇海藻糖激素或其受器基因受到靜默後，卵鞘排出的時間出現顯著性的延遲。 另一方面，該論文研究尚且探討德國蜚蠊昇海藻糖激素具有對抗氧化逆境的功能。該研究使用殺草劑巴拉刈注射到10日齡的雄蟲體內，不僅會造成雄蟲體內的氧化壓力上升並且後來會促使雄蟲死亡。當正常的雄蟲在接受固定濃度的巴拉刈注射後，過半的致死蟲數發生於第4天；然而，額外添加不同濃度的昇海藻糖激素 (10, 40, 80 pmole) 都能夠顯著地延後死亡率的發生，過半的致死蟲數則達第7天以後。此外，巴拉刈的致死作用對於昇海藻糖激素及其受器基因受到靜默而降低表現的雄蟲反而有更劇烈的影響，處理藥劑後的第二天就有半數以上的雄蟲死亡。接下來我進行血淋巴中脂質代謝分析作為昆蟲體內氧化壓力的一項指標。分析的研究結果顯示，將昇海藻糖激素與巴拉刈同時注射到雄蟲體內能夠維持丙二醛 (malondialdehyde) 濃度在正常量的水準；但是對於昇海藻糖激素與其受器基因受到靜默的雄蟲，外加注射的昇海藻糖激素並無法降低氧化逆境的傷害產生，其丙二醛的總量則呈現顯著性地升高。最後，本研究還發現昇海藻糖激素能夠在外加注射巴拉刈的狀態下提高粒線體細胞色素氧化酶第一次單元基因(cytochrome c oxidase subunit I) 與細胞色素基因 (CYP4G19) 的表現量。上述的兩個基因曾經被指出與昆蟲抗氧化能力反應相關，關於昇海藻糖激素抵抗氧化壓力的機制亦在本論文研究中進一步討論。

A hypertrehalosemic hormone (HTH) peptide has been identified in the adipokinetic hormone (AKH) family to induce mobilization of carbohydrate in many insects. The aim of this study was to investigate the functions of hypertrehalosemic hormone in relation to reproduction and antioxidative stress response in the German cockroach Blattella germanica. I have isolated the Blage-HTH and its receptor (Blage-HTHR) genes, and thus studied their function through RNA interference (RNAi) to knockdown their expressions. The conventional role of HTH mediating trehalose homeostasis was observed in both sexes, in which the hemolymph trehalose was significantly reduced by RNAi-mediated knockdown of either Blage-HTH or Blage-HTHR genes. Especially, Blage-HTH regulated the increase of hemolymph trehalose in parallel with the ovarian development in the virgin females. Furthermore, Blage-HTH would assist the oviposition process because the ootheca production was delayed in the virgin females with either Blage-HTH or Blage-HTHR silenced. In addition, I have unveiled another function of Blage-HTH against oxidative stress in B. germanica. Paraquat (PQ), a bipyridilium herbicide used in this study to elicit oxidative stress, causes a detrimental effect on the lifespan of 10-day-old adult males. Co-injection of synthetic HTH peptides at 10, 40, and 80 pmol concentrations significantly reduced the speed of mortality increase, with median survival time attained about 7 days in contrast to 4 days without HTH injection. However, PQ treatment caused a rapid mortality increase, with median survival time attained on 2 days, in the male adults whose Blage-HTH and Blage-HTHR were silenced simultaneously. I further examined the lipid peroxidation as an indicator of oxidative stress in the hemolymph after PQ treatment. Results showed that co-injeciton of HTH peptide maintained a normal level of lipid peroxidation, but it was not rescued by HTH in Blage-HTH and Blage-HTHR knockdown specimens. Finally, Blage-HTH also stimulated the expression of COXI (cytochrome c oxidase subunit I) and CYP4G19 (cytochrome P4504G19) in the fat body under oxidative stress elicited by PQ. The mechanism how Blage-HTH potentiates antioxidant protection has been discussed in the present study.