Comparative phylogenetic analysis of intergenic spacers and small subunit rRNA gene sequences of two microsporidian isolates from Antheraea myllita

Abstract

Two microsporidian isolates extracted from infected tasar silkworms (Antheraea mylitta) collected from forest area in Deoghar district, Jharkhand, India were subjected to PCR amplification using intergenic spacer (IGS) region and small subunit rRNA (SSU-rRNA) gene specific primers followed by cloning and sequencing. The IGS and SSU-rRNA gene sequences were analysed to derive the identity of the microspoidian isolates and establish their phylogenetic relationships. The phylogenetic analysis of test isolates included assessment of variation in sequences and length of IGS and SSU-rRNA genes with reference to 16 different microsporidian sequences. The results proved that IGS sequences have more variation than SSU-rRNA gene sequences. Analysis of phylogenetic trees reveal that both test isolates have very close relationship with each other as well as with three Nosema reference species viz., N. philosamia and N. antheraea isolated from Philosamia cynthia ricini and Antheraea pernyi in China respectively, and N. disstriae isolated from Malacosma disstriae in Canada. The test microsporidian isolates revealed closer relationship with other Nosema reference strains compared to Nosema sp. (NIK-1s_mys) from India. The study results indicate that the IGS or/and SSU rRNA sequence based analysis is suitable and valuable to ascertain phylogenetic relationships between various microsporidian strains/species.

Key words: Microsporidia, Antheraea mylitta, small subunit rRNA, intergenic spacer, phylogenetic relationship.