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Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and k cat 1726 min -1 for glucose, and Km 0.39 ± 0.05 mM and k cat 1976 min -1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and k cat 3293 min -1 for glucose, and Km 1.15 ± 0.11 mM and k cat 3732 min -1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

A new microsporidium was isolated from Histia rhodope Cramer (Lepidoptera, Zygaenidae), a pest of Bischofia javanica BL. in China. The morphology and molecular systematic of this novel microsporidian isolate had been described in this study. The spores were long oval and measured 3.1 × 1.9 μm on fresh smears. Ultrastructure of the spores was characteristic for the genus Nosema: 14-15 polar filament coils, posterior vacuole, and a diplokaryon. The sequenced rRNA gene of this isolate is 4309 bp long. The organization of the rRNA gene is 5'-LSU rRNA-ITS-SSU rRNA-IGS-5S-3', which is similar to that of other Nosema species (such as Nosema bombycis). Phylogenetic analysis based on LSU rRNA gene and SSU rRNA gene both revealed that this novel micorsporidian which isolated from H. rhodope had close relationship to the genus Nosema. Additionally, this isolate can also cause systemic infection of Bombyx mori. So, we should pay attention not only to N. bombycis, but also to other microsporidian (such as Nosema sp. HR) in sericulture in the future.