Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification

of polymorphic DNA: Morphological and ultrastructural characterization S. Nageswara Rao, B. Surendra Nath *, G. Bhuvaneswari, S. Raje Urs Seribiotech Research Laboratory, CSB Campus, Carmelram Post, Kodathi, Bangalore 560 035, India Received 20 July 2006; accepted 2 May 2007 Available online 13 May 2007 Among the microsporidian species described so far, at least 200 have been assigned to the genus Nosema (Sprague, 1981). This seemingly disproportionate number of Nosema species could be due to incorrect identifications in part as well. Many of the earlier studies on microsporidia based on morphology, ultrastructure, life cycle, and hostparasite relationships have resulted in the unnecessary creation of a large number of new Nosema species. The difficulties encountered in proper identification of a Nosema species based on infection, light and electron microscopic examinations are well illustrated by Mercer and Wigley (1987), who could not distinguish several Nosema species they have found in a stem borer, Sceliodes cordalis, from 12 other Nosema species known to infect lepidoptera. Raynaud et al. (1998) and Muller et al. (1999) who failed to identify Encephalitozoon cuniculi, Encephalitozoon intestinalis, and Enterocytozoon bieneusi by light microscopic

examination. However, they found the PCR method to be quite sensitive and useful in the diagnosis and differentiation of microsporidians from specimens infected with more than one microsporidian species. Classification based on ultrastructural differences has been replaced by phylogenetic analysis based on DNA marker profiles (Leipe et al., 1993; Baker et al., 1995; Hartskeerl et al., 1995; Mathis et al., 1997; Hung et al., 1998). Phylogenetic reconstruction based on random amplification of polymorphic DNA (RAPD), and comparison of small subunit ribosomal RNA (SSU-rRNA) sequences have successfully been used to detect and classify various microorganisms (CavalierSmith and Chao, 1996; Dugourd et al., 1996) including microsporidia (Kawakami et al., 1992; Vossbrinck et al., 1993; Baker et al., 1994, 1995; Hung et al., 1998; Raynaud et al., 1998; Hatakeyama et al., 2000; Rao et al., 2004, 2005). Tsai et al. (2003) have reported phylogenetic relationships of different isolates of microsporidia infecting different lepidopteran pests using the RAPD technique. A DNA polymorphism assay based on polymerase chain reaction (PCR) using a single set of primers of arbitrary nucleotide sequence has been described (Welsh and McClelland, 1990; Williams et al., 1990) and used in differentiation studies on a variety of organisms, including microsporidian parasites (Hizer et al., 2002; Pollefeys and

Bousquet, 2003; Tsai et al., 2003). It is easier to generate the nucleotide polymorphisms and establish phylogenetic relationships in microsporidia using RAPD markers than RFLPs and SSU-rRNA sequence analysis, as the latter involves Southern blot hybridization, designing primers, cloning and sequencing, all of which are laborious and relatively expensive. Since the genetic diversity of microsporidians infecting silkworms reared in different parts of India has not been evaluated so far using molecular markers, an attempt has been made to understand the genetic diversity and phylogenetic relationships using the RAPD technique and combining this information with pathological, morphological and ultrastructural characters for the identification of different microsporidian isolates.