The rRNA genes of Nosema species and its applications

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

A Nosema ceranae isolate from the honeybee Apis mellifera

Microsporidiosis (nosema disease) of the honeybee, Apis mellifera, has spread worldwide and caused heavy economic losses in apiculture. We obtained a spore isolate from worker ventriculi of A. mellifera colonies kept on the campus of National Taiwan University and sequenced the ribosomal genes. The entire length of the ribosomal DNA is about 3828 bp and the organization is similar to that of Nosema apis. However, the SSUrRNA, ITS, and LSUrRNA sequences have comparatively low identities with those of N. apis (92, 52, and 89%, respectively) and the SSUrRNA has a 99% identity with Nosema ceranae. These results indicate that this isolate is not N. apis, but N. ceranae. Moreover, the morphological characteristics are identical to those of N. ceranae. These results show that nosema disease of the honeybee, A. mellifera, may not be caused solely by the infection of N. apis

Updating Sacbrood Virus Quantification PCR Method Using a TaqMan-MGB Probe

Sacbrood virus (SBV) is a common honey bee virus disease. SBV variants and strains identified in Asian honey bees, Apis cerana, have created confusion in identifications. Although the regional names indicated the expansions of the virus in new regions, pathogenesis, and genomes of these variants are not distinct enough to be a separate virus species. However, current SBV qPCR methods may not detect newly identified A. cerana SBV variants (Ac SBV) according to the genome sequences. Since these Ac SBV can naturally infect A. mellifera and possibly other hymenopterans, ignorance of Ac SBV variants in detection methods is simply unwise. In this report, we updated the qPCR method based on Blanchard’s design that used conserved regions of VP1 to design a TaqMan method with an MGB (minor groove binder) probe. We tested the method in bees and hornets, including A. mellifera, A. cerana, and Vespa velutina. The updated primers and the probe can match published SBV and Ac SBV genomes in databases, and this updated method has reasonable sensitivity and flexibility to be applied as a detection and quantification method before the discovery of variants with more mutated VP1 gene

A scientific note on detection of honeybee viruses in the darkling beetle (Alphitobius diaperinus, Coleoptera: Tenebrionidae), a new pest in Apis cerana cerana colonies

International audienceNo abstract availabl

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells

RNA viruses are often cited as a significant factor affecting the populations of both domestic honey bees and wild pollinators. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail the in vitro transcription of the viral genome RNA in 8–10 kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of the Chinese variant sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a widely used lepidopteran immortal cell line (Sf9) and honey bee pupae. Furthermore, the negative strand of the clone was detected in both Sf9 cells and honey bee pupae, indicating active infection and replication. However, the transfection of Sf9 cells was observed in only a limited proportion (less than 10%) of the cells, and the infection did not appear to spread to adjacent cells or form infective virions. The injection of honey bee pupae with 2500 ng of the IE1-SBV plasmid resulted in high infection rates in Apis cerana pupae but low rates in A. mellifera pupae, although the dosage was comparatively high compared with other studies using in vitro transcribed viral RNA. Our findings suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, albeit requiring additional optimization. However, this method holds substantial potential for facilitating the production of clones with various sequence modifications, enabling the exploration of viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid form may foster collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately offering a comprehensive approach to managing virus diseases in the future

Transcriptional and physiological responses of hypopharyngeal glands in honeybees (Apis mellifera L.) infected by Nosema ceranae

International audienceNosema ceranae infection induces energetic stress, malnutrition, and precocious foraging in Apis mellifera. This study investigated effects of N. ceranae infection on the gene expression, protein content, and enzyme activity in the hypopharyngeal gland of nurse bees. The 285 differentially expressed genes were found between N. ceranae-infected and control bees using RNA-Seq, and 279 were upregulated and 6 were downregulated in infected bees. The protein level was significantly lower in infected bees than in controls, implying protein dysmetabolism and energetic stress in infected bees. The ELISA test showed that the specific activity of amylase was significantly higher in infected bees than in controls. The elevated enzymic activities were accompanied by an increased expressed level of hemolymph juvenile hormone-binding protein-encoding gene in infected bees, suggesting the possibility of Nosema-infected nurse bees to engage in behaviors that are normally performed by foragers