Studies On The Morphology And Taxonomy Of Nosema Philosamiae And Nosema Phyllobroticae

Microsporidia are obligate intracellular parasites of eukaryotes. Its host ranges from protists to invertebrates and vertebrates. Nearly all insects were susceptible to microsporidia, but over half of them belonged to two orders, Lepidoptera and Diptera, such as silkworm, honeybee and eri-silkworm. Although microsporidia were considered to be eukaryotes, they lack some typicaleukaryotic characteristics including mitochondria, peroxisomes, classical stacked Golgi apparatus, and 80S ribosomes. The microsporidia contain 70S ribosomes, ribosomal subunits (30S and 50S), and rRNAs (16S and 23S) with prokaryotic size, and they have no separate 5.8S rRNA. They also lack ?agella, pilus and 9+2 microtubular structure. Up to now, the genome size has been determined for numerous microsporidian species, and they range from just 2.3 Mbp in Encephalitozoon intestinalis to 19.5 Mbp in Glugea atherinae.Spores of Nosema philosamiae measured approximately 4.1×2.2μm on fresh smears. The complete sequence of ribosomal RNA gene (4,314bp) was obtained by PCR amplification with specific primers and sequencing. Sequences of the large subunit gene (LSU rRNA: 2,497bp), the internal transcribed spacer (ITS:188bp), the small subunit gene (SSU rRNA:1,232bp), the intergenic spacer (IGS: 282bp) and the 5S region (115bp) are also provided. The sequence analysis showed that the organization of the rRNA of N. philosamiae was 5'-LSU-ITS1-SSU-IGS-5s-3'which was similar to the pattern of Nosema bombycis,Nosema spodopterae,Nosema antheraeae. Identity and phylogenetic analysis of the rRNAs andα,β-tubulin gene sequences revealed that N. philosamiae had a close relationship with the Nosema type species, N. bombycis. The resultes confirmed that N. philosamiae was a member of Nosema clade at the aspect of molecular biology.The pebrine of silkworm was not only caused by the microsporidian N. bombycis, but also many microsporidia isolated from wild insects can cause the diseae. Nosema phyllobroticae, a microsporidian parasite from the beetles, Phyllobrotica armta Baly, can infect silkworm and also transmitted transovarially to the progeny eggs. Spores of Nosema phyllobroticae are long-oval in shape, and measure 4.7×2.6μm on fresh smears. The ultrastructural features indicate that the isolate possesses some typical characteristics of the genus Nosema. 13~14 polar filament coils of the polar tube, posterior vacuoles and diplokaryon nuclei were all identified. The rRNA gene of the microsporidian N.phyllobroticae was specifically amplified by PCR, cloning and sequencing. Then the complete sequence of rRNA gene of N. phyllobroticae (4,308bp) was obtained. And the phylogenetic trees of rRNA, LSU rRNA and SSU rRNA gene sequence of N. phyllobroticae were constructed by UPGMA methods of MEGA 4.0 soft. Results indicate that N. phyllobroticae has also a close relationship to N. bombycis, and has the same organization of the rRNA with N. bombycis. At the same time, RNAstructure and RnaViz soft were used for constructing the secondary structure of 5S rRNA of N. phyllobroticae which is following a pattern similar to that of others microsporidia and eukaryotes.Study on the complete sequence of rRNA gene and phylogenetic analysis of N. philosamiae and N. phyllobroticae, could lay a foundation for taxonomic status, establishing of specific diagnosis and phylogenetic relationships of the two isolates with others microsporidia.