台灣假單胞菌與大腸桿菌培養液對蚊子幼蟲毒性研究

本論文分析台灣假單胞菌 (Pseudomonas taiwanensis) 對白線斑蚊 (Aedes albopictus) 幼蟲毒性；台灣假單胞菌於Luria-Bertani培養基培養36小時後，取上清液及菌體進行殺蟲活性試驗。結果顯示斑蚊及家蚊幼蟲對台灣假單胞菌上清液菌均相當敏感，而菌體本身則不具殺蟲活性，顯示台灣假單胞菌殺蟲活性物質是一種外分泌性的代謝產物。而做為對照的大腸桿菌 (Escherichia coli)，其培養上清液對白線斑蚊幼蟲也具有相當毒性，處理1小時內死亡率即可達到100%，大腸桿菌菌體本身亦不具殺蟲活性。將二齡白線斑蚊幼蟲飼養於台灣假單胞菌上清液中2小時，即可造成接近100%的死亡率；而四齡白線斑蚊幼蟲對台灣假單胞菌上清液則較不敏感，但將四齡白線斑蚊幼蟲飼養於台灣假單胞菌上清液中12小時，仍可造成45%的死亡率；飼養24小時後，幼蟲死亡率亦可達100%。雖然前人已經從台灣假單胞菌菌體內(pellet) 得到對果蠅 (Drosophila melanogaster) 與小菜蛾 (Plutella xylostella) 具有殺蟲活性的基因tccC 所產生的毒蛋白群。但是本研究顯示台灣假單胞菌對白線斑蚊幼蟲之殺蟲活性並非源自TccC毒蛋白群，因為經高溫滅菌 (121 0C) 與蛋白酶K降解後，並不會影響台灣假單胞菌培養上清液之殺白線斑蚊幼蟲活性。台灣假單胞菌培養上清液對埃及斑蚊幼蟲及熱帶家蚊也具有相同毒性。台灣假單胞菌培養上清液在經過冷凍乾燥、減壓濃縮乾燥後，其等體積還原液則會失去殺幼蟲活性。分別使用有機溶劑 (正己烷、正丁醇、乙酸乙酯) 萃取分層後，僅有正己烷分層所獲得之水層保有原先毒殺白線斑蚊幼蟲之活性。切片觀察顯示台灣假單胞菌培養上清液處理的白線斑蚊幼蟲腸道組織受損。但以液相層析 (HPLC) 分析台灣假單胞菌培養上清液，並未發現與其殺幼蟲活性相關特殊成份。因此台灣假單胞菌上清液內之可能殺蟲有效物質仍須後續探討。

關鍵字

白線斑蚊；台灣假單胞菌；大腸桿菌；菌液上清代謝物；生物殺蟲劑

並列摘要

This thesis evaluates the mosquitocidal activity of Pseudomonas taiwanensis supernatant. The supernatant derived from P. taiwanensis culture in LB (Luria-Bertani) medium for 36 hours were subjected to mosquitocidal assay against Aedes and Culex mosquitoes. The results show that all mosquito larvae are very sensitive to the supernatant (but not the pellet) of P. taiwanensis, indicating that the mosquitocidal ingredients are extracellular metabolites. The mortality of 2nd instar Ae. albopictus larvae could reach 100% after exposed to the cultural supernatant for 2 hr. The 4th instar larvae exhibited less sensitivity toward P. taiwanensis, however it still showed around 40% mortality after 12 hr exposure to the supernatant. The supernatants (but not the pellet) of Escherichia coli are also demonstrated with significant toxicity to 2nd instar Ae. albopictus larvae, reaching 100% after exposed to the cultural supernatant less than 1 hr. Though an insecticidal gene (tccC) toward Drosophila was identified and cloned from the P. taiwanensis, it is apparently not the responsible toxin for Ae. albopictus larvae, since the mosquitocidal activity is not affect by autoclave at 121 0C for 30 min. However, lyophilization and vacuum evaporation under reduce pressure will abolish the mosquitocidal activity of P. taiwanensis supernatant. The mosquitocidal activity remains in water layer derived from hexane partition after fraction the supernatant with hexane, butanol and ethyl acetate, indicating that the mosquitocidal ingredients are hydrophobic metabolites. Moreover, the anatomical sections showed that the midgut was the major tissue affected by the P. taiwanensis supernatant treatment. However, the high performance liquid chromatography (HPLC) analysis did not show any particular ingredients specifically exist in P. taiwanensis supernatant. The active ingredients of P. taiwanensis supernatant or mechanism responsible for the mosquiticidal activity remain to be solved in the future.