Tasar silk is indigenous to India and tasar culture is a traditional tribal activity in the forests and its adjoining areas, where abundant natural wealth of host plants (Terminalia tomentosa, Terminalia arjuna and Shorea robusta) exists. Tasar silk producing silkworm, Antheraea mylitta is highly heterogenic with several eco-races in varied topographical diverse areas exhibiting diversity in phenotypic characters and forms an integral part of forest eco-system (Rao 2007). 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.

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.

[Fig. 1.] Map of Jharkhand showing the distribution of tropical tasar, A. mylitta in nine geographical forest areas.

[Table 1.] Details of the twenty three microsporidian isolates and type species: their place of collection, host and morphology

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 ocular micrometer under phase contrast microscope and all the measurements are presented in micrometers as mean values of 12 individual observations (Table 1).

Genomic DNA was extracted from the sporoplasm discharged from spores using glass bead method described by Undeen and Cockburn (1989). DNA concentration and quality was determined both by spectrophotometry at 260 and 280 nm and on 0.8% agarose gel, using a known quantity of λDNA (10 ng/μL) as a standard before use in subsequent PCRs. Possibility of host DNA contamination was ascertained using insect mitochondrial primer amplification in PCR. A working solution of DNA (10 ng/μL) was prepared in sterile autoclaved double distilled water.

PCR reactions were performed according to the protocols of Welsh and McClelland (1990) and Williams et al. (1990). A total of 20 RAPD primers (Operon Technologies, Inc, Alameda, CA) were used for PCR amplification. The PCR amplifications were carried out in MJ Research Thermal Cycler PTC-200 (MJ Research Inc. Watertown, MA.) in 20 μL reaction mixture containing 1 x PCR buffer, 200 μM each dNTP’s, 2.5 mM MgCl₂, 0.2 μM of a single primer, 30 ng template DNA and 1 U of Taq DNA polymerase (Thermo Scientific). Amplification reactions were carried out for 35 cycles after an initial denaturation for 3 min at 93°C. Each PCR cycle comprised three steps: denaturation at 93°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 2 min with a final extension of 10 min at 72°C. A negative control without DNA was also run simultaneously in the same thermocycler. The amplified PCR products were size fractionated by electrophoresis on 1.5% agarose gel (Promega corporation, Madison, USA) in 1 x Tris borate EDTA buffer (89 mM Tris, 89 mM Boric acid, 2 mM EDTA, pH 8.0) and gels were stained with ethidium bromide (0.5 μg/mL) for 30 min (Sambrook et al. 1989). A standard molecular weight marker (Mass ruler DNA ladder, Thermo Scientific) was used in each electrophoretic run and UV trans-illuminated gels were photographed by using Gel Documentaion System (Syngene Corporation, Cambridge, U.K.). Experiments were carried out in triplicate on different occasions to verify the reproducibility of markers.

Interpretation of patterns was based on the presence or absence of unequivocally reproducible amplified bands and their size. RAPD markers were scored according to the presence (1) or absence (0) of a band across twenty three isolates of microsporidian; each primer was scored separately. The scoring was repeated three times and reproducible conspicuous bands were only included in the analysis. The total number of fragments amplified, number of poloymorphic fragments scored, and percentage of polymorphic bands were recorded. NTSYS-pc version 2.11T computer program (Applied Biostatistics, Setauket, NY) was used for genetic distance analysis. The data were analyzed using SIMQUAL (similarity for qualitative data) method to generate similarity/genetic distance among different microsporidian isolates using Dice coefficients (Dice 1945) (S = 2N_ab/(2N_ab+N_a+N_b), where N_ab is the number of bands common to lanes a and b, Na is the total number of bands present in a, and Nb is the total number of bands in lane b (Nei and Li 1979). The Dice similarity coefficients were also used to construct a dendrogram using the un-weighted pair group method with arithmetic averages (UPGMA) employing the SAHN (sequential, agglomerative, hierarchical, and nested clustering) module. To evaluate the robustness of obtained UPGMA based dendrogram and their confidence limits, bootstrapping with 1000 replications was performed using the software WINBOOT developed at IRRI, Manila, Philippines (Yap and Nelson 1996). In order to test the genetic variability further, multidimensional scaling of the RAPD data was carried out using the ALSCAL algorithm (SPSS Inc. Chicago, USA). In this method a dissimilarity matrix was created using Euclidean distance and the same was used for the classical Young-Householder multidimensional scaling procedure (Young et al. 1984, Young and Harris 1990).

The different microsporidian isolates identified from diseased tropical tasar silkworm, A. mylitta (Table 1) were characterized using spore morphology. The shape of fresh spore of different microsporidians varied from oval to elongate form measuring 3.80 to 5.10 μm in length and 2.56 to 3.30 μm in width (Table 1). NIK-1s_mys, type species is oval in shape measuring 3.80 μm in length and 2.60 μm in width (Table 1).

Twenty RAPD primers were screened for fingerprinting of the 22 microsporidian isolates along with type species out of which sixteen primers that yielded good amplification were utilized. The amplified products obtained with primer OPW-17 are depicted at Fig. 2. The size of amplified products with different primers ranged from 200 to 3500 bp (Table 2). A total of 308 RAPD fragments were generated with 16 primers of which 97% were polymorphic (Table 2). The primer OPW-8 generated the least number (10) of fragments while OPW-17 generated the highest number (26) (Table 2). Values of genetic distance obtained from each pair wise comparison of RAPD fragments are shown at Table 3. The relationship between 22 isolates and type species as revealed by genetic distance from dice similarity matrix RAPD data varied from 0.059 to 0.980 (Table 3). The genetic distance similarity matrix was least (0.059) between MIJ-1gC and MIJ-3sD while it was highest (0.980) between MIJ-5mD and MIJ-2pD (Table 3). UPGMA based dendrogram utilizing the genetic distance values of RAPD data is presented in Fig. 3. The dendrogram indicated clustering of the different microsporidians into four groups (Fig. 3). The first and second groups contained seven isolates each [Group 1: MIJ-1sG, MIJ-4 mG, MIJ-1gDn, MIJ-1bR, MIJ-1kK, MIJ-2pR and MIJ-3rR from Giridih, Dhanbad, Ranchi and Kharshwan, respectively; Group 2: MIJ-2bG, MIJ-3gG, MIJ-1jD, MIJ-3gWS, MIJ-2 mWS, MIJ-4nWS and MIJ-1cWS collected from Giridih, Deoghar and West Singhbhum, respectively]. The third group clustered four isolates [MIJ-2pD, MIJ-5mD, MIJ-4cD and MIJ-3sD] from a single location viz. Deoghar, while the last group had five isolates [MIJ-1gC, NIK-1s_mys, MIJ-1kDm, MIJ-2dES and MIJ-1cES collected from Chibasha, Mysore, Dumka and East Singhbhum, respectively]. According to geographical location, ten microsporidian isolates from West Singhbhum, East singhbhum and Deoghar clustered together, while rest of the microsporidian isolates clustered randomly along with other microsporidian isolates from different geographical regions (Fig. 3).

[Fig. 2.] Random Amplified Polymorphic DNA (RAPD) banding profiles obtained on 2% agarose gel for the twenty three microsporidian isolates with the primer OPW-17. For the details of 1-23 microsporidians, see Table 1. The lane marked M shows the molecular size marker. The bands marked with arrows are the specific unique bands amplified, which are detailed in Table 4.

[Table 2.] The nucleotide sequences of the primers, number of amplified fragments, fragment size, and number of polymorphic fragments scored using RAPD profiles of twenty three microsporidians DNA in RAPD - PCR

[Table 3.] Dice genetic similarity distance matrix values based on RAPD data between twenty-three microsporidian isolates.

[Fig. 3.] Dendrogram showing genetic relationships among the twenty three microsporidian isolates identified from A. mylitta using UPGMA method. Numbers on each node indicate bootstrap values.

The two-dimensional scaling of the RAPD data, using ALSCAL algorithm based on Euclidean distance matrix, has clearly delineated each of the identified microsporidian from the tasar silkworms as well as from the type species, NIK-1s_mys. (Fig. 4). Of the 22 microsporidian isolates, MIJ-1kDm is found to be closer to NIK-1s_mys, indicating that MIJ-1kDm is similar to the type species and remaining 21 isolates which differed from each other and from type species, were considered to be different variants. The grouping of different microsporidians based on Euclidean distance matrix is very similar as like in the UPGMA based dendrogram (Fig. 4).

[Fig. 4.] Spatial distribution of different microsporidians infecting tasar silkworm, A. mylitta based on the ALSCAL multidimensional scaling method using Euclidean distance matrix. For the details of 1-23 microsporidians, see Table 1.

RAPD analysis also revealed isolate specific amplified products and 14 RAPD primers revealed 45 highly reproducible unique genetic markers. Four bands were specific to the type species, NIK-1s_mys and the remaining 41 bands are obtained to other microsporidian isolates collected from different forest locations (Table 4).

[Table 4.] RAPD-PCR unique genetic markers that distinguish the 23 microsporidians. For the details of 1-23 microsporidians, see Table 1. Sign (？) absent, (+) present, (++) two bands present.

Characterization of different microsporidian isolates from diseased tropical tasar silkworm, A. mylitta using spore morphology and RAPD analysis revealed that the spore of type species (NIK-1s_mys) is similar to the type species N. bombycis maintained at Sericultural Experimentation Station, Tokyo, Japan with GenBank accession number D85503 with an oval shape measuring a length of 3.80 μm and width of 2.60 μm. As Canning et al. (1999) suggested that determination of status of any microsporidian species/isolate should be made against the accession D85503, NIK-1s_mys is included as reference isolate/species in the present study for comparison with the new microsporidian isolates obtained from A. mylitta. The shapes of other isolates range from oval to elongate oval with significant variations in width and length. Takizawa et al. (1975), Sato et al. (1982), Rao et al. (2007) and Nath et al. (2011) reported detailed structure, shape and size of different isolates/species of microsporidians identified from the mulberry silkworm, Bombyx mori which showed significant variations in spore length and width. The constraints for proper identification of microsporidians using morphology and ultrastructural studies are well demonstrated by various researchers (Mercer and Wigley 1987, Raynaud et al. 1998, Muller et al. 1999, Rao et al. 2005, Nath et al. 2011). Molecular markers developed during the past two decades have considerably reduced the problems associated with distinguishing and systematic classification of microsporidians. Initially, Restriction Fragment Length Polymorphisms (RFLPs) served as reliable markers for genetic analysis (Undeen and Cockburn 1989, Didier et al. 1995, Fedorko et al. 1995). Presently, researchers are adopting PCR based techniques among which RAPD technique was found to be sensitive and easier for development of DNA markers (Mathis et al. 1997, Tsai et al. 2003, Rao et al. 2007, Nath et al. 2011).

The RAPD-PCR banding patterns generated using genomic DNA from 23 microsporidian isolates including type species NIK-1s_mys revealed clear polymorphic banding pattern from each of the random primers that delineated 22 microsporidians with good bootstrap confidence values from the type species (NIK-1s_mys). Analysis of the RAPD-PCR banding profile indicated that only MIJ-1kDm isolate is similar to NIK-1s_mys while the remaining 21 microsporidian isolates can be considered as variants. The results suggest that, these specific/unique genetic markers obtained on RAPD-PCR profile could be used as diagnostic tool to differentiate various microsporidian spores. RAPD has been utilized for genetic characterization of different microsporidians infecting humans and silkworms [Mathis et al. (1997), Tsai et al. (2003), Rao et al. (2007) and Nath et al. (2011)].

One of the multivariate approaches of grouping based on the Euclidean distance matrix is the Multidimensional scaling method (Young et al. 1984, Young and Harris 1990). In the present study, pictorial representation of data on different microsporidians using ALSCAL- multidimensional scaling has not only supported the information generated by the UPGMA dendrogram, but it has made the Euclidean distances among the microsporidians more clear. In both instances the groupings were very similar and in the UPGMA, the genetic distance values were used to construct dendrogram using the method of Nei and Li (1979), whereas in ALSCAL- multidimensional scaling the Euclidean method (Young et al. 1984, Young and Harris 1990) was used to obtain similarity matrix. Both methods could discriminate microsporidians isolated from A. mylitta from each other as well as from the type species. Out of 22 isolates identified, ten clustered exactly according their geographical locations. In dendrogram, three groups revealed 12 microsporidian isolates with very different geographical regions clustered randomly which could probable be due to frequent transportation of seed cocoons from one sericulture station to the other for conducting rearing of the tasar silkworms. Grouping pattern of microsporidians isolates supports sympatric speciation in the bio-geographical sericulture area of Jharkhand, India. However, the genetic variation is high among microsporidian isolates collected from this area, which may be due to the obligatory parasitic nature of microsporidian spores.

The present study depicts the simplest approach for identification and discrimination of microsporidian isolates from tropical tasar silkworm, A. mylitta based on RAPD-PCR technique. It also proves that molecular tools like RAPD-PCR analysis that revealed high levels of polymorphism are one of the best approaches with small amounts of genomic DNA as compared to RFLP technique. PCR based fingerprinting technique like RAPD is informative to establish the extent of genetic diversity and phylogeny between different microsporidian isolates available in diverse geographical areas.