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Wazid Hassan and B. Surendra Nath Genetic diversity and phylogenetic relationships among microsporidians isolates from the Indian tasar silkworm 170 171 Materials and Methods Origin of microsporidian isolates. Twenty two microsporidians were collected from individual infected tasar silkmoths of A. mylitta during the survey conducted from 2010 to 2013 in different geographic reserved forests areas (districts of Giridih, Deoghar, Dumka, Dhanbad, Kharshawan, Chaibasha, WestSinghbhum, East-Singhbhum and Ranchi in Jharkhand State, India (Fig. 1 and Table 1). Microsporidian spores were isolated from infected tasar silkmoths by maceration and suspended them in 0.85% NaCl followed by filtration through cheese cloth and centrifugation at 3500 r/min for 10 min. The spore pellet was Percoll purified by gradient centrifugation as described by Undeen and Alger (1971). Each of the purified microsporidian isolates were maintained in vivo in isolation, through per oral inoculation and designated as MIJ-1sG, MIJ-2bG, MIJ-3gG, MIJ-4mG, MIJ-1jD, MIJ-2pD, MIJ-3sD, MIJ-4cD, MIJ-5mD, MIJ-1kDm, MIJ-1gDn, MIJ-1bR, MIJ-2pR, MIJ-3rR, MIJ-1kK, MIJ-1gC, MIJ-1cWS, MIJ-2mWS, MIJ-3gWS, MIJ-4nWS, MIJ-1cES and MIJ-2dES along with type species NIK-1s_mys. The details of microsporidian isolates, places of collection, host, shape and size are presented in Table 1. Measurement of spore length and width. The morphology of purified spores was observed under phase contrast microscope. The length and width of spores were measured according to the method of Undeen and Vavra (1997). Fresh spores were spread in water agar on glass micro-slides and measured using an At present, tasar silk production has increased considerably; but even after there is huge gap in the production and demand. The important reasons for the low production are attributed to traditional rearing on trees in a natural habitat, which exposes the larvae to a number of diseases caused by microbial pathogens apart from the natural calamities. As tasar silkworm rearing is outdoor in nature, it is affected by several diseases viz., microsporidiosis, virosis, bacteriosis and muscardine. The crop loss in tasar culture due to silkworm diseases varies from 40 50% (Sing et al. 2011). The disease caused by the microsporidian, Nosema bombycis is the most devastating in tasar silkworms and inflicts severe cocoon crop loss and passed onto the next generation transovarially. Microsporidia are a diverse group of spore-forming obligate intracellular parasites including more than 1300 formally described species in 160 genera (Wittner and Weiss 1999, Keeling 2009). Microsporidia are unique eukaryotes, which do not possess centrioles, and mitochondrial apparatus, although nuclei are present in distinct number (Vossbrinck and Woese 1986, Vossbrinck et al. 1987). These parasites infect a wide range of invertebrates and vertebrates including insects, fishes, mammals and protists (Wittner and Weiss 1999, Wasson and Peper 2000, Weiss 2001). Large numbers of new microsporidian species are designated based on morphology, ultrastructure, life cycle and host–parasite relationships. Molecular phylogenetic analysis based on DNA marker profiles have largely overcome the problems associated with ultrastructural and phenotype-based classification systems (Baker et al. 1995, Hartskeerl et al. 1995, Mathis et al. 1997, Hung et al. 1998). Random Amplified Polymorphic DNA-PCR assay using a set of primers of arbitrary nucleotide sequences (Welsh and McClelland 1990, Williams et al. 1990) has been described as potential molecular marker system for the analysis of genetic diversity and phylogeny in a wide variety of organisms (Hadrys et al. 1992, Lu and Rank 1996). RAPD technique has been utilized to generate molecular markers for determining the genetic diversity and phylogenetic relationship among Nosema species/strains (Tsai et al. 2003, Rao et al. 2007, Nath et al. 2011). The paper reports the genetic diversity of microsporidians infecting tasar silkworm (A. mylitta), isolated from varied geographical forest locations in the State of Jharkhand (India) using RAPD-PCR technique. Fig. 1. Map of Jharkhand showing the distribution of tropical tasar, A. mylitta in nine geographical forest areas. Int. J. Indust. Entomol. Vol. 29, No. (2), pp. 169-178 (2014) 170 171 Ta bl e 1 . D et ai ls of th e t w en ty th re e m ic ro sp or id ia n iso la te s a nd ty pe sp ec ie s: th ei r p la ce o f c ol le ct io n, h os t a nd m or ph ol og y

international journal of industrial entomology

Genetic Diversity and Phylogenetic Relationships among Microsporidian Isolates from the Indian Tasar Silkworm, Antheraea mylitta, as Revealed by RAPD Fingerprinting Technique