Hexokinase (HXK) is the first key enzyme in the glycolytic pathway and plays an extremely important role in energy metabolism. By searching the microsporidian database, we found a sequence (NBO_27g0008) of Nosema bombycis Nägali, 1857 with high similarity to hexokinase-2, and named it as NbHXK2. The NbHXK2 gene has 894 bp and encodes 297 amino acids with 34.241 kD molecular weight and 5.26 isoelectric point. NbHXK2 contains 31 phosphorylation sites and 4 potential N-glycosylation sites with signal peptides and no transmembrane domain. Multiple sequence alignment showed that NbHXK2 shares more than 40% amino acid identity with that of other microsporidia, and the homology with hexokinase-2 of Nosema tyriae Canning, Curry, Cheney, Lafranchi-Tristem, Kawakami, Hatakeyama, Iwano et Ishihara, 1999, Nosema pyrausta (Paillot, 1927) and Nosema ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 was 89.17%, 87.82% and 69.86%, respectively. Phylogenetic analysis based on the amino acid sequence of hexokinase showed that all microsporidia cluster together in the same clade, and are far away from animals, plants and fungi, and that N. bombycis is closely related to N. tyriae; N. pyrausta; N. ceranae and Nosema apis Zander, 1909. Immunolocalisation with the prepared polyclonal antibody showed that NbHXK2 was mainly distributed in the cytoplasm and plasmalemma in proliferative, sporulation stage and mature spore of N. bombycis. qRT-PCR assay showed that the NbHXK2 expressed at higher level during spore germination and at early stage of proliferation. These results indicate that N. bombycis may use its own glycolytic pathways to supply energy for infection and development, especially germination and in the early stage of proliferation, and acquire energy from the host through certain ways as well.
Microsporidia are intracellular parasites of insects and other higher eukaryotes. The microsporidian Nosema philosamiae Talukdar, 1961 was isolated from the eri silkworm, Philosamia cynthia ricini Grote. In the present study, alpha- and beta-tubulin genes from N. philosamiae were characterized. The identity analysis of nucleotide and amino acid sequences indicated high similarity with species of Nosema Nägeli, 1857 sensu lato (nucleotide sequences, ≥ 96.0%; amino acid sequences, ≥ 99.0%). However, the tubulin genes of N. philosamiae share low sequence similarity with that of N. ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 (strain BRL01) and a Nosema/Vairimorpha species. Phylogenies based on alpha-, beta- and combined alpha- plus beta-tubulin gene sequences showed that N. philosamiae, along with the true Nosema species, forms a separate clade with a high bootstrap value, with N. ceranae BRL01 forming a clade of its own. The results indicated that the alpha- and beta-tubulin sequences may be useful as a diagnostic tool to discriminate the true Nosema group from the Nosema/Vairimorpha group.
Microsporidia are a group of obligate intracellular unicellular eukaryotes that can parasitize a wide variety of other eukaryotes ranging from protists to invertebrates and vertebrates. In this study, we examined the microsporidium Nosema sp. isolated from the mulberry pest, Hemerophila atrilineata Butler, 1881, named herein ''Nosema sp. HA''. The fresh spores were long oval in shape, 3.8 ± 0.4 μm in length and 1.9 ± 0.3 μm in width. Analysis of tissue infection of silkworm, Bombyx mori Linnaeus, 1758, indicated that the midgut, Malpighian tubules, muscle, fat body, silk glands, hemocytes, nerve tissue and gonads of silkworm were infected with Nosema sp. HA. The complete rRNA gene sequence of this microsporidium contained 4 305 base pairs (GenBank Accession JN882299), including the large subunit rRNA (2 492 bp), the internal transcribed spacer (187 bp), the small subunit rRNA (1 232 bp), the intergenic spacer (279 bp) and the 5S region (115 bp). The organization of the rRNA gene is 5'-LSU-ITS-SSU-IGS-5S-3'. Phylogenetic analysis, comparison of sequence identities and the arrangement in the rRNA gene subunits suggested that this isolate is separate from other Nosema species.