Morphology And Phylogenetic Analysis On Endoreticulatus Sp. Zhenjiang And Nosema Sp. MPr

Microsporidia are obligate intracellular parasites of eukaryotes, and could infect insects, fish, rabbits, fur animals and primate, and often cause major economic influence on animal farming. The phylum consists of approximately 160 genera and over 1 300 species. The silkworm disease pébrine was caused by infection of microsporidia and it could transmit through the embryo and food transmission. So all sericulture countries list microsporidia as the only quarantine target in silkworm.In this study the morphological and molecular characteristics of a new isolated microsporidium from the silkworm (Bombyx mori) named Endoreticulatus sp. Zhenjiang were examined. The fresh spores were oval, 2.9±0.2μm in length and 1.2±0.2μm in width. The infectivity of this novel microsporidium in the silkworm was litter weak compared with that of Nosema bombycis, with a median infectious dose (IC50) of 85 363 spores/ml. The complete rRNA cistron has a length of 4 432 base pairs (GenBank Accession No. FJ772431), including the large subunit rRNA (2 460 bp), the internal transcribed spacer (187 bp), the small subunit rRNA (1 254 bp), the intergenic spacer (276 bp) and the 5S region (115 bp). The organization of the rRNA gene is 5′-LSU-ITS-SSU-IGS-5S-3′, which is reverse compared to the organization of most microsporidian rRNA regions. Phylogenetic analysis based on small subunit rRNA sequences showed that this isolate belongs to the genus Endoreticulatus, and is closely related to Glugoides intestinalis. Further, both had a similar reverse arrangement of the rRNA gene. Our study provides another example of a microsporidian species with a novel organization of rRNA genes, demonstrating that the reverse arrangement is exhibited not only by the microsporidian genus Nosema but may also occur in a clade that contains the genera Endoreticulatus and Glugoides.Although microsporidia are spread parasites, only a few Pieris rapae infected with Nosema species have been reported. Here we reported a strain of Nosema species (Nosema sp. MPr) which was isolated from Pieris rapae from Zhenjiang. By a large number of SSU rRNA gene cloning and sequencing, we found that the morphological of Pieris rapae microsporidia of Zhenjiang are various, and with different lengths of SSU rRNA sequences of 1 227 bp, 1 229bp, 1 232bp, 1 233bp, 1 234bp and 1 245 bp, respectively. This is consistent with the results of previous studies. The results based on the cell infection, along with the nucleotide sequences shows that Nosema sp. MPr does not infect BmN cell in vitro and also does not infect silkworm through feeding experiment. The core sequences of rRNA were sequenced and the organization of rRNA gene is 5′-SSU rRNA-ITS-LSU rRNA-3′, which was similar to traditional arrangement. Phylogenetic trees based on SSU rRNA and LSU rRNA gene sequences were constructed by MEGA 4.0 software. The topology shows that Nosema sp. MPr has a close relationship with Nosema genus, and was on the same branch with the second class of Nosema microsporidia. In conclusion, the microsporidium Nosema sp. MPr was classified as a member of Nosema genus.