Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: Morphological and ultrastructural characterizationIntroductionMaterials and methodsMicrosporidian sporesPathogenicity of microsporidian isolatesExamination of infected larval tissuesMode of transmissionScanning electron microscopyTransmission electron microscopyDNA extraction and purificationPCR amplification of the DNA with RAPD primersMolecular data analysisResults and discussionAcknowledgmentsReferencesGenetic diversity and phylogenetic relationships amongmicrosporidia infecting the silkworm, Bombyx mori, usingrandom amplification of polymorphic DNA:Morphological and ultrastructural characterizationS. Nageswara Rao, B. Surendra Nath*, G. Bhuvaneswari, S. Raje UrsSeribiotech Research Laboratory, CSB Campus, Carmelram Post, Kodathi, Bangalore 560 035, IndiaReceived 20 July 2006; accepted 2 May 2007Available online 13 May 2007AbstractRandom amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultra-structural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori.The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to bemore virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmissionin the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8–16coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded atotal of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whosepresence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of bandsharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dicecoefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g,NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were foundto be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microspo-ridian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect poly-morphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in thisprocess have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy. 2007 Elsevier Inc. All rights reserved.Keywords: Microsporidia; Nosema; Silkworm; Bombyx mori; Ultrastructure; Polar filament coils; Genetic diversity; Random amplification of polymo-rphic DNA (RAPD)1. IntroductionMicrosporidia are a group of diverse spore-formingobligatory parasitic amitochondrial protozoans thatinclude more than 1200 species belonging to 143 genera(Weiss and Keohane, 1999; Hoffman et al., 2003). Theseorganisms infect a broad range of invertebrates and verte-brates including insects, fishes, and mammals (Wittner andWeiss, 1999; Wasson and Peper, 2000; Weiss, 2001). ‘Peb-rine’ is the most dreaded disease affecting the mulberry silk-worm, Bombyx mori. It is caused by the microsporidianNosema bombycis Naegeli (Naegeli, 1857) with once devas-tated the silk industry in France during the mid 19th cen-tury. It is thus important to the silk industry.Earlier studies largely relied on the morphology of theparasites particularly the shape and size of spore, variousstages of the life cycle, the length of the polar filament,the number of coils in the polar filament and their angleof tilt, and the organs infected for identification and0022-2011/$ - see front matter  2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2007.05.001*Corresponding author. Fax: +91 080 28439597.E-mail address: [email protected] (B.S. Nath).www.elsevier.com/locate/yjipaAvailable online at www.sciencedirect.comJournal of Invertebrate Pathology 96 (2007) 193–204Journal ofINVERTEBRATEPATHOLOGYtaxonomical studies (Burges et al., 1974; Ball et al., 1981;Sato et al., 1982). Among the microsporidian speciesdescribed so far, at least 200 have been assigned to the genusNosema (Sprague, 1981). This seemingly disproportionatenumber of Nosema species could be due to incorrect identi-fications in part as well. Many of the earlier studies onmicrosporidia based on morphology, ultrastructure, lifecycle, and host–parasite relationships have resulted in theunnecessary creation of a large number of new Nosema spe-cies. The difficulties encountered in proper identification ofa Nosema species based on infection, light and e lectronmicroscopic examina tions are well illustrated by Mercerand Wigley (1987), who could not distinguish severalNosema species they have found in a stem borer, Sceliodescordalis, from 12 other Nosema species known to infect lep-idoptera. Raynaud et al. (1998) and Muller et al. (1999) whofailed to identify Encephalitozoon cuniculi, Encephalitozoonintestinalis, and Enterocytozoon bieneusi by light micro-scopic examination. However, they found the PCR methodto be quite sensitive and useful in the diagnosis and differen-tiation of microsporidians from specimens infected withmore than one microsporidian species. Classification basedon ultrastructural difference s has been replaced by phyloge-netic analysis based on DNA marker profiles (Leipe et al.,1993; Baker et al., 1995; Hartskeerl et al., 1995; Mathiset al., 1997; Hung et al., 1998). Phylogenetic reconstru ctionbased on random amplification of polymorphic DNA(RAPD), and comparison of small subunit ribosomalRNA (SSU-rRN A) sequences have successfully been usedto detect and classify various microorganisms (Cavalier-Smith and Chao, 1 996; Dugourd et al., 1996) includingmicrosporidia (Kawakami et al., 1992; Vossbrinck et al.,1993; Baker et al., 1994, 1995; Hung et al., 1998; Raynaudet al., 1998; Hatakeyama et al., 2000; Rao et al., 2004,2005). Tsai et al. (2003) have reported phylogenetic