Primary study on Nosema bombycis Infecting the Insect of Drosophila

[ Objective ] The aim of this study was to investigate the infectivity of Nosema bombycis to drosophila, which offered a new vision for systematical studies on the infection mechanism of Nosema bombycis, and also provided reference for the bio-control effect of Nosema bombycis. [ Method ] Nosema bombycis was used to feed wild type and mutant drosophila, and the morphological observation of Nosema bombycis in drosophila body fluid was also analyzed by calcofluor white M2R fluorescent staining. [ Result] Nosema bombycis could infect drosophila, and the number of Nosema bombycis in the infected mutant drosophila was higher than that in wild type drosophila. [ Conclusion ] Nosema bombycis can infect drosophila, which provides primary reference for studies on the infectivity of Nosema bombycis to other hosts and also lays a foundation for further study on the infection mechanism of Nosema bombycis.

Comparative Study on the Infectivity and Spore Surface Protein of Nosema bombycis and Its Morphological Variant Strain

A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophilaatrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. Comparativestudies on morphology, infectivity and spore surface protein were conducted. 24Nbh was short and wide, and had asignificant difference (P<0.01) over the Nb spores. The infectivity tests conducted on second instar silkworm larvaeshowed that IC50 of 24Nbh was 1.98104 spores mL-1 and of Nb was 1.72103 spores mL-1, thus indicating that the infectivityof Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The IC50 of spores from silkworm infected with24 Nbh was 6.9 times less than Nb, showing that the infectivity of 24Nbh spores rejuvenated very fast when reinfected tosilkworms, further more, the length and width of such spore was larger than 24Nbh (P<0.01) and smaller than Nb (P<0.05).The SDS-PAGE profiles of Nb and 24Nbh were generally the same, 4 distinct proteins of 12, 17, 30, 33 kDa were obtainedwith difference in quantity. When 120 g of protein was applied for 2D-PAGE, five suspected different proteins withdifference in quantity were observed. These results demonstrate that these differential proteins maybe associated withvariation in infectivity of the spores.

Phagocytic Uptake of Nosema bombycis (Microsporidia) Spores by Insect Cell Lines

Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involves its polar tube acting as a needle-syringe system. However, recent studies show microsporidian spores can also gain access to host cells by phagocytosis. The present study investigated the phagocytic uptake process of causative agent of the pebrine disease, Nosema bombycis, in several insect cell lines. We observed KOH-treated spores and cold-storaged spores can be easily uptaken by all the studied cell types 4 h post inoculation. In contrast, large numbers of freshly recovered spores remained in the culture medium. To further investigate the intracellular fates of KOH-treated spores and cold-storaged spores, electron and fluorescence microscopy were performed. No intracellular germination or subsequent parasite development were observed. Intracellular spores can be detected in host cells by polyclonal antibody 7 d post inoculation, suggesting phagocytized N. bombycis could not be digested by these non-professional phagocytes. Our results suggest that, phagocytic uptake of N. bombycis spores might represent a defense mechanism of the host cells and the intact spore wall barrier enable freshly recovered spores to keep resistance to this mechanism.

Mitochondrial pyruvate dehydrogenase E1 of Nosema bombycis:A marker in Microsporidian evolution

Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this phylum. We present here the mitochondrial pyruvate dehydrogenase El （PDH, including PDHα and PDHβ） of the microsporidian Nosema bombycis, the pathogen of silkworm pebrine. Compared with PDH of microsporidian Encephalitozoon cuniculi and Antonospora locustae, both subunits are eonscrced. The phylogeny indicated that both subunits are mitochondrial. The syntenic maps revealed the subunits organization of NbPDH is distributed in different scaffolds, similar to that of EcPDH but different with AIPDH, and the relationship between phylogeny tree and organization of PDH suggest that the AlPDH subunits organization is the ancestral style of microsporidia, and through the genome evolution, the reshuffling of the chromosome of microsporidia occurred, the adjacent style of ALPDHE1 organization changed, and the two subunits separated and located to different chromosomes in E. cuniculi. For N. bombycis and N. ceranae, they locate to different scaffolds. In order to determine NbPDH subcellular localizations, we prepared the polyclonal antibodies against NbPDH prokaryotic fusion proteins, and adopted the colloidal gold immunological electron microscopy, the expression signals of NbPDH were observed in spores however, the subcellular localization were not definited. In general, through comparison of three mierosporidian PDH molecular phylogeny, subunits organization in chromosomes, localization indicated that PDH is an interesting marker in microsporidia evolution