臺灣博碩士論文加值系統

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.

目錄
中文摘要……………………........…………………………… 1
英文摘要……………………………………..………………………… 3

第壹章、前言………………………...………………………… 5

第貳章、材料與方法……………………….…………..……… 17

第參章、結果………………………………...………………… 27

第肆章、討論…………………………..……………………… 33

參考文獻……………………………………….………………………… 45
附錄………………………………………………..…………… 57

柯俊成、陳秋男、王重雄。 2002。 菸草粉蝨 (Bemisia tabaci species complex) 分類學綜述。 台灣昆蟲 22: 307-341。
Akman, L. & Aksoy, S. 2001. A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies. Proc. Natl Acad. Sci. USA 98: 7546-7551 .
Akman, L., Yamashita, A., Watanabe,, H., Oshima, K., Shiba, T., Hattori, M. & Aksoy S. 2002. Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nature Genetics 32:402-407 .
Aksoy, S. 1995. Molecular analysis of the endosymbiont of tsetse flies: 16S rDNA and overexpression of a chaperonin Insect Mol. Biol 4:29-32
Andersson, S. G. & Kurland, C. G. 1998. Reductive evolution of resident genomes. Trends Microbiol. 6: 263-268 .
Baumann, L., Baumann, P. & Thao, M. L. 1999. Detection of messenger RNA transcribed from genes encoding enzymes of amino acid biosynthesis in Buchnera aphidicola (endosymbiont of aphids). Curr. Microbiol. 38: 135-136 .
Baumann, L., Thao, M.L., Funk, C. J., Falk, B. W., Ng, J. C. K. & Baumann, P. 2004. Sequence analysis of DNA fragments from the genome of the primary endosymbiont of the whitefly Bemisia tabaci. Curr. Microbiol. 48: 77-81 .
Brown, J. K., D. R. Frohlich, & R. C. Rosell. 1995. The sweetpotato or silverleaf whiteflies: biotypes of Bemisia tabaci or a species complex? Annu. Rev. Entomol. 40: 511-534 .
Brynnel, E. U., Kurland, C. G., Moran, N. A. & Andersson, S. G. 1998. Evolutionary rates for tuf genes in endosymbionts of aphids. Mol. Biol. Evol. 15: 574-582 .
Buchner, P. Endosymbiosis of Animals with Plant Microorganisms (Interscience, Inc., New York, 1965)
Charles, H. & Ishikawa, H. 1999. Physical and genetic map of the genome of Buchnera, the primary endosymbiont of the pea aphid Acyrthosiphon pisum. J. Mol. Evol. 48: 142-150 .
Charles, H., Mouchiroud, D., Lobry, J., Goncalves, I. & Rahbe, Y. 1999. Gene size reduction; in the bacterial aphid endosymbiont, Buchnera. Mol. Biol. Evol. 16: 1820-1822 .
Clark, M. A., Moran, N. A. & Baumann, P. 1999. Sequence evolution in bacterial endosymbionts having extreme base compositions. Mol. Biol. Evol. 16: 1586-1598 .
Clark, M. A., Moran, N. A., Baumann, P. & Wernegreen, J. J. 2000. Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence. Evolution Int. J. Org. Evolution 54: 517-525 .
Clark, M. A., Baumann, L., Thao, M. L., Moran, N. A. & Baumann, P. 2001. Degenerative minimalism in the genome of a psyllid endosymbiont. J. Bacteriol. 183: 1853-1861 .
Cock, M.J.W. 1986. Bemisia tabaci, a literature survey on the cotton whitefly with an annotated bibliography. CAB IIBC, Silwood Park, UK. 121 pp.
Cock, M.J.W. 1993. Bemisia tabaci, an update 1986~1992 on the cotton whitefly with an annotated bibliography. CAB IIBC, Silwood Park, UK. 78 pp
Cole, S. T., Eiglmeier, K., Parkhill, J. & 42 other authors. 2001. Massive gene decay in the Leprosy bacillus. Nature 409: 1007-1011 .
Combell, B. C.,J. D. Steffen-Campbell, and R. J. Gill. 1996. Origin and radiation of whiteflies: an initial molecular phylogenetic assessment, p. 29-51. In : D. Gerling and R. T. Mayer (Eds.), Bemisia 1995: Taxonomy, Biology, Damage, Control and Magnagement. Intercept, Andover, United Kingdom.
Costa, H. S., Westcot, D. M., Ulman, D. M., Rosell, R., Brown, J. K. & Johnson, M. W. 1995. Morphological variation in Bemisia tabaci endosymbionts. Protoplasma 189: 194-202 .
Costa, H. S., Toscano, N. C. & Hennberry, T. J. 1996. Mycetocyte inclusion in the oocytes of Bemisia argentifolii (Homoptera: Aleyrodidae). Arthopod Biology 89: 694-699 .
Costa, H. S.,Henneberry, T. J. & Toscano, N. C. 1997. Effects of antibacterial materials on Bemisia argentifolii (Homoptera: Aleyrodidae) oviposition, growth, survival, and sex ration. J. Econ. Entomol. 90: 333-339 .
Dale, C., Young, S. A., Haydon, D. T. & Welburn, S. C. 2001. The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc. Natl Acad. Sci. USA 98: 1883-1888 .
Dale, C., Plague, G. R., Wang, B., Ochman, H. & Moran, N. A. 2002. Type III secretion systems and the evolution of mutualistic endosymbiosis. Proc. Natl Acad. Sci. USA 99: 12397-12402 .
Dasch, G., Weiss, E. & Chang, K. in Bergy’s Manual of Systematic Bacteriology Vol. 1 (eds Holt, J. & Krieg, N.) 811–833 (Williams & Williams, Baltimore, Maryland, 1984).
De Barro, P. J. & Driver, F. 1997. Use of RAPD PCR to distinduish the B biotype from other biotypes of Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae). Aust. J. Entomol. 36: 149-152 .
De Barro, P. J. & Hart, P. J. 2000. Mating interactions between two biotypes of the whitefly, Bemisia tabaci(Hemiptera:Aneyrodidae) in Australia. Bull. Entomol. Res. 90: 103-112 .
Douglas, A. E. 1989. Mycetocyte symbiosis in insects. Biol. Rev.Camb. Phil. Soc. 64: 409-434 .
Douglas, A.E. 1998. Nutritional interaction in insect-microbial symbioses. Annu Rev Entomol 43: 17-37 .
Fares, M. A., Barrio, E., Sabater-Munoz, B. & Moya, A. 2002a. The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol. Biol.Evol.19: 1162-1170 .
Fares, M. A., Ruiz-Gonzalez, M. X., Moya, A., Elena, S. F. & Barrio, E. 2002b. Endosymbiotic bacteria: groEL buffers against deleterious mutations. Nature. 417: 398 .
Filichkin, S. A., Brumfield, S., Filichkin, T. P., and Young, M. J. 1997. In vitro interactions of the aphid endosymbiont symL chaperonin with barley yellow dwarf virus. J. Virol. 71: 569-577.
Frohlich, D. R., Torres-Jerez, I. & Bedford, I. D. 1999. A phylogeograohical analysis of the Bemisia tabaci sepecies complex based on mitochondrial DNA markers. Mol. Ecol. 8: 1683-1691 .
Fukatsu, T., Kaoru, W. & Yuji, S. 1998. Specific detection of intracellular symbiotic bacteria of aphids by oligonucleotide-probed in situ hybridization. Entomol. Zool. 33: 461-472 .
Funk, D. J., Wernegreen, J. J. & Moran, N. A. 2001. Intraspecific variation in symbiont genomes: bottlenecks and the aphid–Buchnera association. Genetics 157: 477-489 .
Gil, R., Sabater-Munoz, B., Latorre, A., Silva, F. J. & Moya. 2002. A. Extreme genome reduction in Buchnera spp.: toward the minimal genome needed for symbiotic life. Proc. Natl Acad. Sci. USA 99: 4454-4458 .
Gil, R., Silva, F.J., Zientz, E., Delmotte,F., Gonzalez-Candelas,F., Latorre,A., Rausell,C., Kramerbeek,J., Gadau,J., Hoelldobler,B., van Ham,R.C.H.J., Gross,R. & Moya,A. 2003. The genome sequence of Blochmannia floridanus: Comparative analysis of reduced genomes. Proc. Natl. Acad. Sci. 16: 9388-9393 .
Goebel, W. & Gross, R. 2001. Intracellular survival strategies of mutualistic and parasitic prokaryotes. Trends Microbiol. 9: 267-273 .
Hentschel, U., Steinert, M. & Hacker, J. 2000. Common molecular mechanisms of symbiosis and pathogenesis. Trends Microbiol. 8: 226-231 .
Ishikawa H. 1982. Isolation of the intracellular symbionts and partial characterizations of their RNA species of the elder aphid, Acyrthosiphon magnoliae. Comp. Biochem. Physiol. 72B: 239-247 .
Kirk, A. A.,L.A.Lacey,J.K. Brown, M.A. Ciomperlik, J. A. Goolsby, D, C. Vacek, L. E. Wendel, and B. Napompeth. 2000. Variation in the Bemisia tabaci species complex (Hemiptera: Aleyrodidae) and its natural enemies leading to successful biological control of Bemisia biotype B in the USA. Bull. Entomol. Res. 90: 317-327 .
Lambert, J. D. & Moran, N. A. 1998. Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria. Proc. Natl Acad. Sci. USA 95: 4458-4462 .
Lawrence, J. & Roth, J. in Organization of the Prokaryotic Genome (ed. Charlesbois, R.) 263-289 (ASM Press, Washington, DC, 1999) .
Ling, V. S., Jeremy, M. F., George, T., Midori, O., Claudio, B., Barton, E. S. & Scott, O. 2001. Determination of Wolbachia genome size by pulsed-field gel electrophoresis. J. Bacteriol. 183: 2219-2225 .
Ludwig, W., O. Strunk, S. Klugbauer, N. Klugbauer, M. Weizenegger, L. Neumaier, M. Bachleitner, and K. H. Schleifer, 1998. Bacterial phylogeny based on comparative sequence analysis. Electrophoresis. 19: 554-568 .
Milkman, R. & Bridges, M. M. 1993. Molecular evolution of the Escherichia coli chromosome. IV. Sequence comparisons. Genetics 133: 455-468 .
Mira, A., Ochman, H. & Moran, N. A. 2001. Deletional bias and the evolution of bacterial genomes. Trends Genet. 17: 589-596 .
Mira, A. & Moran, N. A. 2002. Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria. Microb. Ecol. 44: 37-143 .
Moran, N. A. 1996. Accelerated evolution and Muller’s ratchet in endosymbiotic bacteria. Proc. Natl Acad. Sci. USA 93: 2873-2878 .
Moran, N. & Telang, A. 1998. Bacteriocyte-associated symbionts of Insects. Bioscience 48: 295-304 .
Moran, N. A. & Baumann, P. 2000. Bacterial endosymbionts in animals. Curr. Opin. Microbiol. 3: 270-275
Moran, N. A. & Wernegreen, J. J. 2000. Lifestyle evolution in symbiotic bacteria: insights from genomics. Trends Ecol. Evol. 15: 321-326 .
Moran, N. A. & Mira, A. 2001. The process of genome shrinkage in the obligate symbiont Buchnera aphidicola. Genome Biol. 2, RESEARCH0054.
Moran, N. A. 2002. Microbial minimalism: genome reduction in bacterial pathogens. Cell. 108: 583-586 .
Morin, S., Ghanim, M., Zeidan, M., Czosnek, H., Verbeek, M. & Johannes F. J. M van den Heuvel. 1999. A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus. Viology 256: 75-84 .
Morin, S., Ghanim, M., Sobol, I. & Czosnek, H. 2000. The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system. Virology 276: 404-416 .
Munson, M.A., Baumann, P., Clark, M. A., Baumann, L., Moran, N. A., Voegtlin, D. J. & Compbell, B. C. 1991. Evidence for establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. J. Bacteriol. 173: 6321-6324 .
Ochman, H. & Wilson, A. C. 1987. Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J. Mol. Evol. 26: 74-86.
Ochman, H. & Moran, N. A. 2001. Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis. Science 292: 1096-1099 .
Ohta, T. 1973. Slightly deleterious mutant substitutions in evolution. Nature 246: 96-98 .
Ohtaka, C. & Ishikawa, H. 1993. Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. J. Mol. Evol. 36: 121-126 .
Palacios, C. & Wernegreen, J. J. 2002. A strong effect of AT mutational bias on amino acid usage in Buchnera is mitigated at high expression genes. Mol. Biol. Evol. 19:1575-1584 .
Rispe, C. & Moran, N. A. 2000. Accumulation of deleterious mutations in endosymbionts: Muller’s ratchet with two levels of selection. Am. Nat. 156: 424-441 .
Rosell, R. C., Costa, H. S., Coombs, M. T., Frohlich, D. R. & De Barro., P. 2004. Phylogenetic characterization of primary endosymbionts associated with diverse whitefly species. International congress of entomology XXII, Brisbane Queensland Australia.
Sandstrom, J., Talang, A. & Moran, N. A. 2000. Nutritional enhancement of host plants by aphids — A comparison of three aphid species on grasses. J. Insect Physiol. 46: 33-40 .
Sato, S. & Ishikawa, H. 1997. Expression and control of an operon from an intracellular symbiont which is homologous to the groE operon. J. Bacteriol. 179: 2300-2304 .
Scott, O., Rosanna, G., Angela, M. E. C., Timothy, L. K. & Hugh, M. R. 1992. 16S rRNA phylogenetic analysis of the bacyerial endosymbionts associated with cytoplasmic imcompatibility in insect. Proc. Natl Acad. Sci. USA 89: 2699-2702 .
Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y. & Ishikawa, H. 2000. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81-86 .
Spaulding, A. W. & von Dohlen, C. D. 1998. Phylogenetic characterization and molecular evolution of bacterial endosymbionts in psyllids (Homoptera: Sternorrhyncha) Mol. Biol. Evol. 15: 1506-1513 .
Steinert, M., Hentschel, U. & Hacker, J. 2000. Symbiosis and pathogenesis: evolution of the microbe–host interaction. Naturwissenschaften 87: 1-11 .
Swofford, D. L. 1998. PAUP*, Phylogenetic analysis using parsimony (*and other methods), version 4.0b4a, Sinaur Associates, Sunderland, MA.
Tamas, I., Klasson, L., Canback, B., Naslund, A. K., Eriksson, A. S., Wernegreen, J. J., Sandstrom, J. P., Moran, N. A. & Andersson, S. G. E. 2002. 50 million years of genomic stasis in endosymbiotic bacteria. Science 296: 2376-2379 .
Thao, M. L., Gullan, P. J. & Baumann, P. 2002. Secondary (-Proteobacteria) endosymbionts infect the primary (-Proteobacteria) endosymbionts of mealybugs multiple times and coevolve with their hosts. Appl. Environ. Microbiol. 68: 3190-3197 .
Thao, M. L., Baumann, L., Hess, J. M., Falk, B. W., Ng, J. C. K., Gullan, P. J. & Baumann, P. 2003. Phylogenetic evidence for two new insect – associated Chlamydia of the family Simkaniaceae. Curr. Microbiol. 47: 46-50 .
Thao, M. L. & Baumann, P. 2004a. Evolutionary relationships of primary prokar yotic endosymbionts of whiteflies and their hosts. Appl. Environ. Microbiol.70: 3401-3406 .
Thao, M. L. & Baumann, P. 2004b. Evidence for multiple acquisition of Arsenophonus by whitefly species (Sternorrhyncha: Aleyrodidae). Curr. Microbiol. 48: 140-144 .
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, & D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acida Res. 25: 4876-4882 .
Tsutomu Tsuchida, Ryuichi Koga & Takema Fukatsu. 2004. Host plant specialization governed by facultative symbiont. Science 303: 1989 .
van den Heuvel, J. F. J. M., Verbeek, M., & van der Wilk, F. 1994. Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae. J. Gen. Virol. 75: 2559- 2565 .
van den Heuvel, J. F. J. M., Bruyeire, A., Hogenhout, S. A., Ziegler-Graff, V., Brault, V., Verbeek, M., van der Wilk, F., & Richards, K. 1997. The all-beta-protein. J. Biol. Chem. 267: 16829-16833 .
Von Dohlen, C. D., Kohler, S., Alsop, S. T. & McManus, W. R. 2001. Mealybug -proteobacterial endosymbionts contain -proteobacterial symbionts. Nature 412: 433-436 .
Weber, H. 1935. Der Bau der Imago der Aleurodinen. Zooligica 33: 1-71 .
Wernegreen, J. J. & Moran, N. A. 1999. Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes. Mol. Biol. Evol. 16: 83-97 .
Wernegreen, J. J., Ochman, H., Jones, I. B. & Moran, N. A. 2000. Decoupling of genome size and sequence divergence in a symbiotic bacterium. J. Bacteriol. 182: 3867-3869 .
Wernegreen, J. J., Lazarus, A. B. & Degnan, P. H. 2002. Small genome of Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies irreversible specialization to an intracellular lifestyle. Microbiology 148: 2551-2556 .
Wernegreen, J. J. 2002. Genome evolution in bacterial endosymbionts of insects. Nature 3: 850-861 .
Zchori-Fein, E., & J. K. Brown. 2002. Diversity of prokaryotes associated with Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Ecology and Population Biology. 95: 711-718