台灣貓蚤簇蟲感染貓蚤之生物學及其應用

Abstract

摘要 貓蚤 (Ctenocephalides felis) 是世界性害蟲，可攜帶多種人畜疾病病原，每年全世界也花費數十億的金錢防治與根除貓蚤。而簇蟲 (gregarines) 是原生動物類的寄生蟲，分類上屬於頂複合器門 (Apicomplexa)，通常簇蟲生存在海生或陸生無脊椎生物的腸腔中，簇蟲大多具有宿主專一性的生活史。因此，研究貓蚤的簇蟲與其宿主的影響與關係，或許可以應用於跳蚤的生物防治。本論文研究目標為研究台灣的貓蚤簇蟲 (Steinina ctenocephali (Ross)) 在貓蚤的生活史、檢視不同時期的簇蟲形態、調查簇蟲與貓蚤族群動態變化、最後再探討貓蚤簇蟲對宿主的影響。我們於台北市動物之家以蚤梳法調查貓狗的外寄生蟲3年，記錄跳蚤種類、性別與數量，並觀察感染貓蚤簇蟲的情形，分析影響簇蟲感染的因子，部份感染的簇蟲用來進行分類與生活史研究，另外也解剖感染簇蟲的貓蚤，取其簇蟲粹取DNA進行SSU rRNA基因PCR後進行序列分析，建構簇蟲分子系統學。感染貓蚤部份以微小盒飼養，收取簇蟲卵囊體，再以此進行感染試驗，探討貓蚤簇蟲對貓蚤的影響。結果顯示，貓蚤簇蟲完全在貓蚤腸道中發育，首先，非活動期的卵囊體可以在環境中存活很久，甚至長達140天，當卵囊體被幼蟲食入後，厚壁的卵囊體會在腸道中釋出孢子體。這些孢子體會先變成胞內時期，並在貓蚤吸血後而開始發育。剛羽化的貓蚤在吸血3-5天後會出現發育中的孢子體，並利用頂器附著在腸壁細胞上。行有性生殖時，兩隻簇蟲會前後結合，形成配子體。當配子體成熟後會再釋出其中數以千計的卵囊體於貓蚤腸道中。另外，我們增幅出1778 bps簇蟲的SSU rRNA基因，與其他簇蟲進行最大相似度與Bayesian分析，發現貓蚤簇蟲與Monocystis agilis Stein, Prismatospor aevansi Ellis 及Syncystis mirabilis Schneider相近，但與Gregarina屬不在同一單系群中。再加上形態的差異，顯示Ross當初將貓蚤簇蟲置於Gregarina屬是有問題的。依形態結果，貓蚤簇蟲應該隸屬於Steinina屬。其次三年調查結果顯示，炎熱的季節簇蟲感染率比寒冷的季節高；雌貓蚤簇蟲感染率高於雄蚤；狗身上的貓蚤比較容易感染簇蟲。最後，感染試驗顯示貓蚤簇蟲不具有生物防治貓蚤的潛能，因為簇蟲感染對貓蚤存活率與羽化率沒有任何的影響。但是，有趣的是高濃度卵囊體處理組，貓蚤的發育時間有顯著縮短的現象。

Abstract The cat flea, Ctenocephalides felis, is a worldwide pest able to transmit pathogens of humans and animals so that billions of dollars worldwide annually are devoted to their control and/ or eradication. Gregarines are protozoan endoparasite members of the phylum Apicomplexa living in the internal cavities of marine and terrestrial invertebrate. The group is considered to be represented by species which are mostly host-specific with monoxenous life cycle. Thus, once gregarines were found in cat fleas in Taiwan we decide gain insight into the relationship. These data may represent the baseline information regarding how to use it as potential biocontrol agents against cat flea. This study therefore aims to study life history, morphology, taxonomy and molecular systematics, population dynamics and host-parasite relationship (i.e., effects) of Steinina ctenocephali (Ross) (Gregarine, Apicomplexa) isolated from cat fleas in Taiwan. Fleas were collected in Taipei Animal Shelter, by combing cats and dogs over a 3-year period. Under stereomicroscope S. ctenocephali-infected fleas were: i) dissected and S. ctenocephali’s stages, either microphotographed “in vivo”, stored for conventional DNA isolation (PCR, cloning, and sequencing), or electronic microscopy processing and ii) left alive and reared in microcells for recovery of S. ctenocephali oocysts. Sex of the infected fleas, type of host (i.e., dogs and cats), prevalence and intensity of gregarine infection were recorded. The whole life cycle of S. ctenocephali occurred exclusively in the flea’s gut. The oocysts (infective stage) can keep alive in the environment up 140 days. Once ingested by the flea larvae, sporozoites are released in the gut of flea larvae. Sporozoites then differentiate into trophozoites in the newly emerged fleas. Trophozoites are found attached firmly to gut of the flea by a cup-shaped epimerite with flattened bottom as support. A pair of trophozoites are then caudo-frontally associated, and then gametocysts are formed. Mature gametocysts release hundreds of oocyst into the gut of the flea as the starting point of the entire life cycle. Partial SSU rRNA gene of S. ctenocephali was amplified (approximately 1,778 bp) and analyzed for relationship whitin and among gregarines species. The S. ctenocephali appears to be closely related to Monocystis agilis Stein, Prismatospora evansi Ellis and Syncystis mirabilis Schneider but not in the same monophyly with Gregarina. Morphological and molecular data are in perfect agreement supporting the relocation of Gregarina ctenocephali Ross to the genus Steinina Lèger and Duboscq. Over three years study we found that S. ctenocephali was more prevalent in warm seasons than the cold one, female’s fleas were more attacked by gregarines, and fleas living on dogs are more prone to be infected with gregarines compared to those on cats. Gender and host preference were evident as female’s fleas were more vulnerable to be infected by gregarines, and fleas living on dogs invariably were found to be infected by more S. ctenocephali compared to cats. The artificial infection of S. ctenocephali on cat fleas indicated that this gregarine may not be an appropriate biocontrol agent. However, developmental time of flea was shortened when exposed to high dose of oocysts, which may represent an interesting, but previsouly undiscovered, case involving adaptative host-manipulation by S. ctenocephali.