台灣銀葉粉蝨懷菌細胞於超微形態上及基因體之研究

Abstract

銀葉粉蝨 (Bemisia argentifolii) 寄主範圍廣泛，歷年來有逐漸擴增的趨勢。此蟲以吸食植物韌皮部汁液為食，其所需營養成分，必須由共生菌提供一些必需胺基酸，相同地共生菌亦須仰賴寄主才能存活，因此在演化上，銀葉粉蝨與共生菌是生死與共關係。銀葉粉蝨體內之共生菌存在特化細胞之胞質內，此細胞稱為懷菌細胞 (mycetocyte)。由於銀葉粉蝨寄主範圍廣泛，各類植物所提供養分必然不同，且皆不足以提供銀葉粉蝨所需，所以共生菌在此類寄主域的宿主內所扮演的角色更加重要。因此本研究利用顯微鏡確定並觀察其懷菌細胞的位置及構造，並以專一性引子28F及1495R增幅初級共生物之 16S rDNA，作為螢光原位雜合之探針，及建構粉蝨總科及胸喙亞目初級內共生菌間的親緣關係。更建立出純化銀葉粉蝨初級內共生菌的方法，初步定出銀葉粉蝨初級內共生菌的基因體大小。銀葉粉蝨成蟲之懷菌細胞散佈在腹部末端，懷菌細胞是屬大型的細胞，核位於中央，細胞核內異染色質 (Heterochromatin) 均勻分布。初級內共生菌充斥於胞質內，形狀不定，切片常會有一類核構造。共生菌與胞質間形成一明顯地電子疏鬆空間，未見明顯膜狀的構造包圍，在胞質內亦有分裂的現象，懷菌細胞的胞質稀疏的介於懷菌細胞內。懷菌細胞內的初級內共生菌呈現自體捲曲，造成菌體的切面可觀察到痕紋。此初級共生菌16S rDNA序列，作為螢光原位雜合法之探針觀察銀葉粉蝨雌成蟲的腹部切片，可見懷菌細胞及腹側前端卵原細胞的胞質均有初級內共生菌的存在，除了佐證初級共生菌的分布，同時提出了卵母細胞在尚未發育時已有游離的初級共生菌傳染的分子證據。銀葉粉蝨初級內共生菌的基因體大小約為1,020 k.b.p.，相較其他含有初級內共生菌的昆蟲，其初級內共生菌基因體較大。此結論與16S rDNA 序列所繪出之胸喙亞目初級內共生菌親緣關係樹相符，而較大的基因體同時會留存較多基因，也解釋了銀葉粉蝨近年來寄主範圍擴大的現象。

The whitefly, Bemisia argentifolii, has a host rang of wide plants and tends to expand gradually. They are a phloem-sap feeder. The nutritional composition of host plants lacks essential amino acids that they need. The nutrient deficiency of their diet is supplemented by endosymbionts that are mutually symbiotic for their survivals. Therefore, the relationship between B. argentifolii and its primary endosymbionts are obligate relationship. The endosymbionts live in the cytoplasm of a specialized cell, called mycetocyte. Due to a wide host range of B. argentifolii, the nutritional composition supplied by different host plants may be varied that makes endosymbionts to play a more important role on nutrient supplementation. In my study, we examined and localized the mycetocyte first and then to reveal the structure of mycetocyte by light and electron microscopies, and amplified 16S rDNA with a specific primer set, 28F and 1695R, for FISH (fluorescence in situ hybridization) probe preparation to examine the distribution of primary endosymbiots. The phylogenetic tree was constructed based on primary endosymbiont’s 16S rDNA sequences to find the relationship of our whitefly with the species of the suborder Sternorrhyncha. Finally, we tried to purify the primary endsymbiont’s genomic DNA from whitefly and to estimate their genome size. Mycetocytes of the B. argentifolii are giant cells that disperse in the hemocoelum at the posterior part of abdomen. The cells possess a nucleus contained homogenously distributed heterochromatin and are filled with pleomorphic primary endosymbiont in cytoplasm, some of primary endosymbiont showed a pseudonucleus in section. An obvious electron-lucent space between primary endosymbionts and cytoplasm was found and led to the perforate cytoplasm of mycetocyte, no obvious membrane bound primary endosymbionts to separate the cytoplasm of mycetocyte. Primary endosymbionts in the cytoplasm of mycetocytes and a mycetocyte in developing oocyte of the female adult of B. argentifolii could be clearly observed by paraffin serial sections and FISH. In addition, the FISH-positive reaction was also found in the tissue of oogonium. The genome size of primary endosymbionts is about 1,020 k.b.p. which is larger than that of other species of the suborder Sternorrhyncha. This also was confirmed by the phylogenetic analysis with the species of the suborder Sternorrhyncha and also suggested that this insect needs more genes for their wide and expanding host range.