The full length of ribosomal RNA (rRNA) genes of N. bombycis has been examined and presented in this thesis, and the DNA sequence data has been submitted (4,301 bp, GenBank Accession No. AY259631). The organization of N. bombycis rRNA genes is unique in arrangement, 5′- LSUrRNA- ITS- SSUrRNA- IGS- 5S rRNA -3′. By sequencing the rRNA gene of the closely related species, N. spodopterae, its rRNA genes organization follows a similar pattern to N. bombycis. The secondary structures of the N. bombycis LSU and SSUrRNA genes were constructed and compared with those of other microsporidia. The secondary structures of N. bombycis and N. spodopterae rRNAs showed few differences between those of other known microsporidia. This finding implies that the rRNA genes organization might be an important characteristic of the members in genus Nosema.
Recently, N. ceranae has been reported causing nosema disease in Apis mellifera which was considered infected by N. apis only. Furthermore, the ratio of N. ceranae to other pathogens causing nosema disease in some areas is quite high. The full length rRNA genes of N. ceranae has also been examined and found identical to those of N. apis. Further sequencing the rRNA genes, the rRNA organization of N. ceranae is found to be 5′- 5S rRNA- IGS- SSUrRNA- ITS- LSUrRNA- 3′, and the orientation of 5S rRNA is reversed of LSU and SSUrRNA. However, it is not found in related species. Comparing the rRNA sequences of the isolates from A. mellifera and A. ceranae, it showed no difference in phylogeny, and it may represent no differentiation between the isolates from different hosts. Moreover, the N. ceranae isolates from different areas of the world were obtained through abroad researchers, and these isolates showed little difference in IGS phylogenetic analyses. It might imply that no isolation between these isolates. Recent researches showed that there may be more than two species causing honeybee nosema disease, and it raised the questions about the accuracy of records that attributed the Nosema species infection of A. mellifera. Therefore, a multuplex PCR diagnosis method that could discriminate N. apis and N. ceranae was established in this study. It could discriminate the pathogen within one PCR and prevent the misidentification of species in the nosema disease researches. Furthermore, it could be applied in a large scale survey to clarify the distribution and interaction of different pathogens causing nosema disease of Apis spp.口試委員會審定書………………………………………………………………….. i
誌謝…..……..……………………………………………………………………….. ii
中文摘要…………………………………………………………………………….. iii
Abstract……..……………………………………………………………………….. v
General Introduction ...……………………………………………………………1
Chapter 1 A novel organization and complete sequence of ribosomal DNA gene of Nosema bombycis 13
1.1 Introduction ...…………………………………………………….. 14
1.2 Materials and methods ...………………………………………….. 16
1.3 Results ……………………………………………………………. 18
1.4 Discussion ………………………………………………………... 22
References ……...…………………………………………………….. 27
Chapter 2 Complete sequence and gene organization of the Nosema spodopterae rRNA Gene 35
2.1 Introduction ………………………………………………………. 36
2.2 Materials and methods ……………………………………………. 36
2.3 Results and discussion ……………………………………………. 37
References ……...…………………………………………………….. 41
Chapter 3 A Nosema ceranae isolate from the honeybee Apis mellifera 45
3.1 Introduction ………………………………………………………. 46
3.2 Materials and methods ……………………………………………. 47
3.3 Results ……………………………………………………………. 50
3.4 Discussion ………………………………………………………... 53
References ……...…………………………………………………….. 58
Chapter 4 The unique rDNA organization of Nosema ceranae and comparison of the isolates from different areas 65
4.1 Introduction ………………………………………………………. 66
4.2 Materials and methods ……………………………………………. 67
4.3 Results ……………………………………………………………. 69
4.4 Discussion ...……………………………………………………… 71
References ……...…………………………………………………….. 76
Chapter 5 Molecular diagnosis method for nosema disease 82
5.1 Introduction ………………………………………………………. 83
5.2 Materials and methods ……………………………………………. 84
5.3 Results ……………………………………………………………. 87
5.4 Discussion ………………………………………………………… 89
References ……...…………………………………………………….. 93
Appendix ….……………….…………………………………………. 10
