Nosema ceranae, a microsporidian, has been given much attention in recent years as it has been linked with Colony Collapse Disorder (CCD), which leads to the sudden deaths of honey bee colonies. It has been described that many pathogenic organisms secrete virulence factors in order to hijack its host`s cellular functions, but in most cases the underlying mechanisms of this process still remains to be deciphered. Cornman et al. (2009) have identified in N. ceranae a list of putative effector proteins (called secretome) destined to be secreted into the host, and I have taken this list for further investigation using a bioinformatical and experimental approaches. The principal aim of this project was to generate a N. ceranae ORFeome for genes predicted to be secreted, elucidate the function of effector candidates important for N. ceranae biology and/or pathogenicity, as well as to investigate any interactions between N. ceranae proteins and its host utilising two eukaryotic model organisms, budding yeast, S. cerevisiae, and fruit fly, D. melanogaster. A library of S. cerevisiae strains expressing N. ceranae proteins was generated utilising the Gateway® technology, and phenotypic and localisation screens were undertaken to investigate the N. ceranae secretome. Two N. ceranae ORFs, NcORF-15 (NcORF-02039) and NcORF-16 (NcORF-01159) encoding a putative thioredoxin and a hexokinase, respectively, were subjected to yeast complementation assays in order to assess their catalytic activity. NcORF-15, the putative thioredoxin, was able to rescue the sensitive phenotype of S. cerevisiae Δtrx2 under oxidative stress, whereas NcORF-16, the putative hexokinase, did not complement YSH7.4-3C, a triple knockout lacking hexokinase activity. A third N. ceranae effector candidate NcORF-4 (NcORF-00654), a putative proteasome subunit, was investigated for its nuclear localisation and protein interactions in both S. cerevisiae and D. melanogaster.

4 Summary Altogether, the six most common bee viruses which infect the honey bee (Apis mellifera) were monitored in the territory of the Czech Republic between 2006 and 2009. Parallel infections of viruses (DWV, ABPV and BQCV) in bee adults and parallel co- infection of viruses with fungal diseases caused by Nosema apis and Nosema ceranae were confirmed by PCR tests. A new sensitive method of detection of the originator of the American foulbrood (Paenibacillus larvae) from bee debris was developed for the practical use of detection of AFB disease in bee populations. Various approaches for the extraction of spores from bee debris and lyses of spores were compared. The sensitivity of PCR tests for the presence of Paenibacillus larvae in debris was compared with the classic cultivation method. The PCR method for the detection American foulbrood was further studied and developed to be more efficient. A new method, based on a matrix-like sample re-arrangement and a use of pooled samples, has been developed for testing 1000 samples in 35 PCR reactions. Another goal was to develop a robust and fast screening method for American foulbrood based on the cultivation test using paper sheets RIDA®COUNT with a specific cultivation medium, specific selection conditions for Paenibacillus larvae and chromogen visualization...