Analysis Of Microsporidian Pathogen From Bumblebees Based On SSUrRNA Sequences

Microsporidia are widespread intracellular parasites that infect both vertebrate and invertebrate hosts. Bumblebees are the main pollinator for facilities farming in China, but the distribution and prevalence of microspodidia pathogen in Chinese bumblebees had not been studied deeply. Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. Multiple genera of microsporidian parasites are known to infect the individual insect hosts. Yet closely related host species may nevertheless harbour different microsporidia (Nosema) species. Based on this situation, it might therefore be expected that different microsporidia species parasitise different Bombus species instead of only Nosema bombi. In this study, the genetic diversity of microsporidia in Bombus species was documented across four different locations of China by using SSUrRNA gene sequencing, PCR-RFLP analysis and secondary structure construction. The results were as follows:(1) Bumblebees were widely infected by microsporidia.1008 bumblebees of 26 species from Gansu, Qinghai, Sichuan and Inner Mongolia were detected by the analysis of SSUrRNA gene sequences. The results showed that 210 individuals of 16 species were infected by microsporidia and infection rate was 20.8%, the highest infection rate was found in Bombus festivus (75.0%) and Bombus sibiricus (68.6%), while the lowest infection rate was in Bombus rufofasciatus (1.9%). Furthermore, the highest infection rate was found in bumblebees from Inner Mongolia (50.0%), the second was from Sichuan (22.9%), the lowest was from Gansu (2.1%) and Qinghai (7.7%).(2) Multiple Nosema infected Chinese bumblebees, including Nosema bombi, Nosema ceranae and the Nosema thomsoni which had been found in Tortricidae of Lepidoptera insects. It can be concluded that bumblebees were infected widely by N.bombi and N.ceranae and the microsporidia from other hosts.(3) A rapid method to differentiate microsporidia species was developed based on PCR-RFLPs of partial SSUrRNA PCR products. The fragment was amplified with the primer pair SSUrRNA-f1/SSUrRNA-rlb and digested with the enzyme Afa I, the microsporidia species were identified by the agarose gel electrophoresis analysis.(4) Comparison of SSUrRNA sequences to other microsporidia, phylogenetic analyses and secondary structure construction showed that all these microsporidia from bumblebees were the genera Nosema, and distinct from the "true Nosema" species. All of these microsporidia were within species diversity. The N.cerane isolated from bumblebees identical with that from honey bees, and they showed the same insertion, GATT, after the 116th base of SSUrRNA sequences with 1264 bp length. N.bombi A and N.thomsoni A were highly similar to previously published of N.bombi (AY008373) and N.thomsoni (EU219086), respectively, and the similarity were>99%. The specie of Nosema sp.-1 was about 97.1% identical with N.bombi A, and there were 14 variable sites between Nosema sp.-2 and N.thomsoni A. Nosema sp.-1 and Nosema sp.-2 may be the subspecies of N.bombi and N.thomsoni, respectively.