Among the non-mulberry (vanya) silkworms, eri silkworm, [Samia cynthia ricini, Donovan (Lepidoptera: Saturniidae)] is amenable for domestication and exhibits multivoltine nature with polyphagous feeding habit. Eri silk mostly confined to the Brahmaputra valley of Assam, India in the tribal inhabited districts and other North Eastern states of the country, significantly contributes to Indian commercial silk production. Like mulberry silkworm, eri silkworms are prone to several virulent and infectious diseases and pests. However, they are comparatively more resistant to the diseases and pests than other silkworm types.

Muga silkworm [Antheraea assamensis, Helfer (Lepidoptera: Saturniidae)] is an economically important insect in India. A. assamensis is an endemic species prevalent in the Brahmaputra valley. Muga culture for the people of Assam is part of their culture, tradition and customs, rather than a profitable professional. Presently, about 30,000 families in Assam are directly associated with muga culture. Muga silkworm is very prone to several virulent and infectious diseases and pest. The germs and symptoms of disease are more or less similar to those other silkworm species diseases. All the major pathogenic microbes cause disease in muga silkworm and the most common among them are Pebrine, Grassarie, Flacherie, and Muscardine. Pebrine is caused by Nosema sp. In pebrine disease, silkworm stops feeding resulting in unequal size larvae, they become it sluggish and die. The dead larvae turn black due to secondary bacterial infection.

Microsporidia are a diverse group of obligate intracellular eukaryotic parasites with 1300 described species in 160 genera approximately (Wittner and Weiss 1999, Keeling 2009). Microsporidia are distinctive eukaryotes, which do not have centrioles and mitochondrial apparatus, although, nuclei are present in distinct number (Vossbrinck and Woese 1986, Vossbrinck et al. 1987). Earlier microsporidia were classified in the kingdom Protista. However, recent molecular phylogenetic analysis using various genes viz., α-tubulin, β-tubulin and Hsp-70, suggest that microsporidia are more closely related to fungi (Hirt et al. 1997, Keeling 2003). Microsporidians infect a wide range of invertebrates and vertebrates including insects, fishes, mammals and protists (Wittner and Weiss 1999, Wasson and Peper 2000, Weiss 2001).

Classification of microsporidians based on ultrastructural differences have been replaced by molecular phylogenetic analysis based on DNA marker profiles (Baker et al. 1995, Hartskeerl et al. 1995, Mathis et al. 1997, Hung et al. 1998). The Inter simple sequence repeats (ISSR), (Zeitkiewicz et al. 1994) and Random amplified polymorphic DNA (RAPD), (Williams et al. 1990) were identified as potential molecular marker systems. The ISSR and RAPD-PCR markers are successfully been used to generate molecular markers, to create genetic diversity and phylogenetic relationship among different microsporidians identified from different silkworms (Tsai et al. 2003, Rao et al. 2005, 2007, Nath et al. 2011, Hassan and Nath 2014 ).

ISSR primers that amplify regions between SSRs (referred as inter simple sequence repeats or ISSRs) are very useful for detecting genetic polymorphisms in microsatellite and intermicrosatellite loci and have been found to be a novel technique for fingerprinting and thus differentiating, closely related individuals (Zietkiewicz et al. 1994). The usefulness of ISSR genetic markers has been well established by various researchers (Nagaoka and Oghihara 1997, Fang et al. 1997, Raina et al. 2001, Reddy et al. 1999, Vijayan 2004, Rao et al. 2005). The ISSR-PCR strategy is especially attractive because it does not need sequence information for primer synthesis, enjoying the advantage of random markers.

In Random Amplified Polymorphic DNA-PCR assay a set of primers of arbitrary nucleotide sequences been used (Welsh and McClelland 1990, Williams et al. 1990), which have 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).

So far, no research works are reported on the molecular characterization of microsporidian isolates infecting the Indian muga (A. mylitta) and eri (S. c. ricini) silkworms, with special reference to vast bio-geographical forest areas in different districts of Assam State, India. The present study was undertaken to establish genetic characterization of 10 different microsporidian isolates infecting Indian non-mulberry silkworm, A. assamensis and S. c. ricini using ISSR and RAPD-PCR markers assay.

Ten microsporidians were originally collected from the diseased individual A. assamensis and S. c. ricini silkmoths during 2010 to 2013 in nine locations belonging to six geographic regions covering different traditional muga and eri culture reserved forest areas in the districts of Jorhat, Dhemaji, Darrang, kamrup, Korbi Anglong, and Lakhimpur in Assam, India (Fig. 1). The microsporidian spores were isolated from infected muga and eri 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 purified by Percoll gradient centrifugation (Undeen and Alger 1971). Each of the purified microsporidian isolates were maintained in vivo in isolation, through per oral inoculation and designated as MIA-7mJr, MIA-8mDm, MIA-9mMd, MIA-10mKp, MIA-1eBr ,MIA-2eBr, MIA-3eDj, MIA-4eLr, MIA-5eDu, MIA-6eTr and the type species is designated as NIK-1s_mys. The details of microsporidian isolates, places of collection, host, shape and size are presented in Table 1.

[Fig. 1.] Map of Assam showing the distribution of non-mulberry silkworm Muga (A. assamensis) and Eri (S. c. ricini) in six biogeographical areas.

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

The morphology of purified microsporidian spores was observed using phase contrast microscope. The length and width of microsporidian spores were measured according to the method of Undeen and Vavra (1997). The fresh mature 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 sporoplasms discharged from spores using the glass bead method (Undeen and Cockburn 1989). DNA concentration and quality was determined by spectrophotometry at 260 and 280 nm and on 0.8% agarose gel visualization, using a known quantity of λDNA (10 ng/μL) as a standard before use in subsequent PCRs. The possibility of host DNA contamination was checked using insect mitochondrial primers.

The protocol of Zietkiewicz et al. (1994) was followed with minor modifications. 20 ISSR primers from primer set 9 (Biotechnology Laboratory, University of British Columbia, Vancouver, B.C.) were tested for PCR amplification, of which 14 primers (11 di, 2 tri, and 1 penta-, nucleotides) which were high polymorphic and reproducible observations were used for PCR amplification. These primers were mostly 15 to 18 mers (Table 2). The PCR amplification was carried out in 20 μL of reaction volume, containing 1 x PCR buffer, 30 ng of template DNA, 200 μM of each dNTP’s, 2.5 mM MgCl₂, 100 pM of a single primer and 1 U of Taq DNA polymerase. Samples were amplified on a DNA thermal cycler (MJ Research Inc., Watertown, Mass.). After initial denaturation at 94°C for 2 min, 35 cycles of 30 s denaturation at 94°C, 30 s annealing at 50°C and a 2 min extension at 72°C were performed before a final extension of 10 min at 72°C and subsequent cooling at 4°C. The ISSR amplification PCR products were mixed with bromophenol blue gel loading dye and were size fractionated by electrophoresis on 2.0% 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 (Thermo Scientific, USA) was used in each electrophoretic run and the UV-transilluminated gels were photographed by using Gel Documentation System (Syngene Corporation, Cambridge, UK). Three replicate experiments were carried out to verify the reproducibility of the markers on different occasions.

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

RAPD-PCR reactions were performed according to the protocols of Welsh and McClelland (1990) and Williams et al. (1990). Twenty different 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. The RAPD amplification PCR products were mixed with bromophenol blue gel loading dye and were size fractionated by electrophoresis on 1.5% agarose gel. RAPD amplified fragments similarly processed further like ISSR.

Analysis of the patterns was based on the presence or absence of unambiguously reproducible amplified bands and their size. ISSR and RAPD markers were scored according to the presence (1) or absence (0) of a band across ten isolates of microsporidians; each primer was scored separately. The banding scoring were repeated three times and only the reproducible conspicuous bands were included in the analysis. The total number of fragments amplified, the number of polymorphic fragments scored and the percentage of polymorphic bands were documented. The NTSYS-pc version 2.11T computer program (Applied Biostatistics, Setauket, NY) was used for genetic distance analysis. The ISSR and RAPD data were analyzed using SIMQUAL (similarity for qualitative data) method to generate genetic distance values 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, N_a is the total number of bands present in a and N_b is the total number of bands in lane b) (Nei and Li 1979). The Dice similarity coefficients were then used to generate dendrograms using the un-weighted pair group method with arithmetic averages (UPGMA) employing the SAHN (sequential, agglomerative, hierarchical and nested clustering) module. To check the robustness of the obtained UPGMA based dendrograms and their confidence limits, bootstrapping with 1000 replications was performed using the WINBOOT software (Yap and Nelson 1996). In addition, the genetic variability further tested by multidimensional scaling of the ISSR and RAPD data was carried out using the ALSCAL algorithm (SPSS Inc. Chicago USA). The dissimilarity matrixes were created using Euclidean distance and the same was used for the classical Young-Householder multidimensional scaling procedure in this method (Young et al. 1984, Young and Harris 1990).

The different microsporidian isolates identified from diseased A. mylitta and S. c. ricini silkworm, (Table 1) were characterized using spore morphology. The shape of mature spore of four muga microsporidians found elongated oval from measuring 3.90 to 4.90 μm in length and 2.66 to 2.90 μm in width. Similarly, six eri microsporidians also found elongated oval from measuring 3.80 to 4.05 μm in length and 2.60 to 3..05 μm in width (Table 1). The shape of NIK-1s_mys, a type species is oval measuring 3.80 μm in length and 2.60 μm in width (Table 1).

Genomic DNA from 11 different microsporidian isolates were used to generate ISSR-PCR amplification patterns. Twenty ISSR primers were tested for PCR amplification with all 10 newly identified microsporidian isolates along with a type species. Out of 20 primers, fourteen primers produced good amplification products. They were used for ISSR markers analysis. A majority of 11 primers annealed to dinucleotide repeats, 2 annealed to tri- and 1 to penta nucleotide repeats. Very high degree of polymorphism was detected with all 14 ISSR primers. Total 178 fragments were scored from the fourteen ISSR primers. Out of 178 fragments, 175 (98%) were polymorphic (Table 2). The ISSR-PCR amplified fragments profile generated with ISSR-827 primer shown in Fig. 2A clearly shows the extent of polymorphism among the different microsporidian isolates. The total number of ISSR markers varied in different isolates with different primers. Some of the bands were common to all isolates and the rest were present only in specific isolates. Almost all 11 microsporidian isolates had different ISSR profiles. The size of the amplified fragments ranged from 700 to 3000 bp (Table 2). The ISSR-PCR fingerprinting patterns of 11 microsporidian isolates with various ISSR primers were used to calculate genetic distance values with Dice similarity coefficient method (Dice, 1945). The relationship among the 11 microsporidian isolates, as revealed by genetic similarity calculated from ISSR data, varied from 0.385 to 0.941 (Table 3A), suggesting significant variability among the microsporidians. The lowest value of Dice similarity coefficient (0.385) was found between the MIA- 6eTr and MIA-7mJr isolates. The highest similarity coefficient (0.941) was found between MIA-1eBr and MIA-2eBr (Table 3A). The genetic similarity values were used for constructing the dendrogram using the un-weighted pair group method with arithmetic averages (UPGMA) method (Fig. 3A). The obtained dendrogram grouped 11 microsporidian isolates in to two major (A and B) clusters. Cluster A included four isolates: MIA-7mJr, MIA-8mDm, MIA-9mMd, and MIA-10mKp, isolated from muga silkworm and collected from four district i.e., Jorhat, Dhemaji, Darang and Kamrup respectively. Cluster B, consisted seven isolates viz., MIA-1eBr, MIA-2eBr, MIA-3eDj, MIA-4eLr, MIA-5eDu, and MIA-6eTr isolated from eri silkworm and collected from Korbi Anglong, Lakhimpur, Jorhat, and Dhemaji and type species NIK-1s_mys.

[Fig. 2A.] Inter simple sequence repeat (ISSR) banding profiles obtained on 2% agarose gel for the eleven microsporidian isolates with the primer ISSR-827. The lane marked M shows the molecular size marker.

[Table 3.A] Dice genetic similarity distance matrix values based on ISSR data among eleven microsporidian isolates

[Fig. 3A.] Dendrogram constructed from ISSR data showing genetic relationships among the eleven microsporidian isolates using UPGMA method. Numbers on each node indicate bootstrap values.

Twenty RAPD primers were screened for fingerprinting of the 10 newly identified microsporidian isolates along with type species out of which sixteen primers that yielded good amplification were utilized. The amplified products obtained with primer OPW-6 are depicted at Fig. 2B. The size of amplified products with different primers ranged from 350 to 2900 bp (Table 2). Total 196 RAPD fragments were generated with 16 primers of which 95% were polymorphic (Table 2). Values of genetic distance obtained from each pair wise comparison of RAPD fragments are shown at Table 3B. The relationship between 10 isolates and type species as revealed by genetic distance from dice similarity matrix RAPD data varied from 0.083 to 0.938 (Table 3B). The genetic distance similarity matrix was least (0.083) between MIA-6eTr and MIA-7mJr while it was highest (0.938) between MIA-1eBr and MIA-2eBr (Table 3B). UPGMA based dendrogram utilizing the genetic distance values of RAPD data is presented in Fig. 3B. The dendrogram indicated clustering of the different microsporidians into two groups (A and B). Group A contained four isolates i.e., MIA-7mJr, MIA-8mDm, MIA-9mMd, and MIA-10mKp. All isolates infect muga silkworm and collected from four different districts i.e., Jorhat, Dhemaji, Darang and Kamrup. The B group contained seven isolates viz., MIA-1eBr, MIA-2eBr, MIA-3eDj, MIA-4eLr, MIA-5eDu, and MIA-6eTr isolated from eri silkworm and collected from four different districts in Assam, India and type species NIK-1s_mys isolated from Mysore, Karnataka, India (Fig. 3B).

[Fig. 2B.] Random amplified polymorphic DNA (RAPD) banding profiles obtained on 1.5% agarose gel for the eleven microsporidian isolates with the primer OPW-6. The lane marked M shows the molecular size marker.

[Table 3. B] Dice genetic similarity distance matrix values based on RAPD data among eleven microsporidian isolates

[Fig. 3B.] Dendrogram constructed from RAPD data showing genetic relationships among the eleven microsporidian isolates using UPGMA method. Numbers on each node indicate bootstrap values.

The two-dimensional scaling of the ISSR and RAPD data, using ALSCAL algorithm based on Euclidean distance matrix, has clearly delineated each of the newly identified microsporidian from the muga and eri silkworms as well as from the type species, NIK-1s_mys (Fig. 4A and 4B). Of the 10 microsporidian isolates, two eri microsporidians MIA-5eDu and MIA-6eTr are found to be little closer to NIK-1s_mys, indicating that eri microsoridians are slightly similar to the type species and the muga isolates which differed 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. 4A and 4B).

[Fig. 4A.] Spatial distribution of eleven different microsporidians based on the ALSCAL multidimensional scaling method using Euclidean distance matrix with ISSR data. Details of microsporidians isolates are e1=MIA-1eBr, e2=MIA-1eBr, e3= MIA-3eDj, e4= MIA-4eLr, e5=MIA-5eDu, e6=MIA-1eTr, m1=MIA-7mJr, m2=MIA-8mDm, m3=MIA-9mMd, m4=MIA-10mKp and s1=NIK-1s_mys.

[Fig. 4B.] Spatial distribution of eleven different microsporidians based on the ALSCAL multidimensional scaling method using Euclidean distance matrix with RAPD data. Details of microsporidians isolates are e1=MIA-1eBr, e2=MIA-1eBr, e3= MIA-3eDj, e4= MIA-4eLr, e5=MIA-5eDu, e6=MIA-1eTr, m1=MIA-7mJr, m2=MIA-8mDm, m3=MIA-9mMd, m4=MIA-10mKp and s1=NIK-1s_mys.

The identified microsporidian isolates were characterized using spore morphology and PCR based ISSR and RAPD fingerprinting. The spore of type species, NIK-1s_mys is oval in shape and size measuring 3.80 (length), 2.60 (width) (Table 1). NIK-1s_mys is similar to the type species N. bombycis maintained at Sericultural Experimentation Station, Tokyo, Japan with GenBank accession number D85503. Canning et al. (1999) suggested the determination of the status of new microsporidian isolates should be made against D85503. Hence, NIK-1s_mys is included as reference species in the this study for comparing new microsporidian isolates identified form A. assamensis and S. c. ricini. Earlier, researchers had used the small differences in microsporidian spore size, as an indication of genus. However, small differences in spore size could not be considered as a taxonomic character. The spore size for a given species may vary with the host size (Brooks and Cranford 1972) and is affected by temperature (Maddox and Sprenkel 1978, Medeiros et al. 2004) age of the host and the medium in which they are measured (Malone and Wigley 1981, Mercer and Wigley 1987). In this study, we found muga microsporidians were slightly bigger than eri microsporidians (Table 1) which support Brooks and Cranford (1972) finding.

The genomic DNA from ten microsporidian isolates was used to generate ISSR and RAPD-PCR amplification patterns. The constructed both dendrograms clearly revealed that clustering of 11 microsporidian isolates based on their host silkworm viz., four muga microsporidians [MIA-7mJr, MIA-8mDm, MIA-9mMd, and MIA-10mKp] separated in a separate group while six eri microsporidians [MIA-1eBr, MIA-2eBr, MIA-3eDj, MIA-4eLr, MIA-5eDu, and MIA-6eTr] were separated in other grouped along with a type species. This indicates all the eri microsporidians had close phylogenetic relationship with type species NIK-1s_mys. The dendrograms showed clear separation of all 10 novel microsporidian isolates from each other with a good bootstrap value. In order to distinguished the micirosporidian isolates from each other, further analysis of dendrograms suggest that two eri microsporidian isolates [MIA-5eDu and MIA-6eTr] look slightly similar with type species, NIK-1s_mys.

Even the multidimensional scaling of the ISSR and RAPD data, using the ALSCAL algorithm on Euclidean distance matrix has clearly separated the microsporidians from each other and type species, NIK-1s_mys (Fig. 4A and 4B). The multidimensional scaling method is one of the multivariate approaches of grouping based on the Euclidean distance matrix (Young et al. 1984, Young and Harris 1990). It anticipates being more informative about differentiating distinct and closely related isolates. The use of pictorial representation of data using ALSCAL- multidimensional scaling has not only supported the information generated by the UPGMA dendrogram, but it has made the Euclidean distances among different microsporidians more clear. In both illustrations the grouping were almost similar and it is important to note that in the UPGMA the genetic distance values was 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. The results from both methods gave almost similar patterns of grouping; in both methods microsporidians isolated from A. assamensis and S. c. ricini were discriminated from each other and from type species as well (Figs. 3A, 3B, 4A, and 4B). The grouping pattern of newly identified microsporidians supported their sympatric speciation origin in the bio-geographical sericulture area of Assam, India.

The ISSR fingerprinting results of Rao et al. (2005) showed that, the dinucleotide repeats (AG)_n, (GT)_n; trinuclotide repeats (ATG) _n, (CTC)_n, (GTT)_n were most abundant in the genome of different microsporidian species isolated from the mulberry silkworm, Bombyx mori. Based on the di-, tri- nucleotide amplification results, it is clear that the different microsporidian isolates identified from the A. assamensis and S. c. ricini, are different from the microsporidians identified from the mulberry silkworms (B.mori).

ISSR and RAPD profiles in the present study clearly delineated the 10 microsporidian isolates with good bootstrap confidence values from type species, NIK-1s_mys and confirming the capability of ISSR and RAPD markers to discriminate the different microsporidian isolates (Fig. 3a, b). Rivera et al. (1995), Mathis et al. (1997), Bretagne et al. (1997), Gur-Arie et al. (2000), Shivaji et al. (2000), Sreenu et al. (2003), Tsai et al. (2003), Rao et al. (2005, 2007), Kumar et al. (2007), Nath et al., (2011), Hassan et al.(2014) used ISSR and RAPD markers for genetic characterization and identification of various bacteria, microsporidia and fungi. Thus, the present study forms the first report on molecular characterization of microsporidian isolates from muga and eri silkworm based on ISSR and RAPD-PCR markers assay. The ISSR and RAPD-PCR are one of the simplest and quickest marker systems with high reproducibility, including the virtue of its unique efficiency in distinguishing even closely related organisms and is important for proper identification of different microsporidian isolates. The study confirms that molecular tools including ISSR and RAPD markers analysis are alternative and facilitate more useful genetic diversity studies of different microsporidians infecting various organisms, since this technique requires only a small amount of genomic DNA and can produce high levels of polymorphism.

The study inferred that the ten newly identified microsporidians from muga and eri silkworms differed in their spore morphology and the ISSR and RAPD PCR profiles indicating genetic variability among them. The high level of polymorphism realized from this study further proves the efficacy of ISSR and RAPD markers assay.