Comparative phylogenetic analysis of intergenic spacers and small subunit rRNA gene sequences of two microsporidian isolates from Antheraea myllita

1 Molecular Pathology Division, Seribiotech Research Laboratory, Central Silk Board, CSB Campus, Carmelram Post, Kodathi, Bangalore – 560035, Karnataka, India. 2 Central Tasar Research and Training Institute, Central Silk Board, Piska-Nagri Ranchi-835 303, Jharkhand, India. 3 Central Sericultural Germplasm Resources Centre, Central Silk Board, P.B. 44, Thally Road, Hosur 635109, Tamil Nadu, India.

Haines and Shorea robusta Roxb.Tasar silkworm rearing generates substantial rural employment in India.However, rearing of Antheraea mylitta often causes infectious diseases like microsporidiosis, virosis, bacteriosis and muscardine.Among these, the most devastating is microsporidiosis caused by the microsporidian Nosema sp.causing severe cocoon crop loss (Singh, 2011).Microsporidia are a diverse group of spore-forming obligate intracellular parasites that include more than 1300 described species under 160 genera (Corradi and Keeling, 2009).They infect a wide range of invertebrates and vertebrates including insects, fishes and mammals (Wittner and Weiss, 1999;Weiss, 2001).They have the smallest genomes among eukaryotic organisms and cause a variety of important medical, agricultural, veterinary, sericulture and ecological impacts (Keeling and Fast, 2002).The ultrastructural and phenotypebased classification systems faced several problems that were largely overcome through ribosomal DNA (r-DNA) sequence analysis (Baker et al., 1995;Hung et al., 1998).Several studies have been attempted to classify microsporidian species and strains through amplification and sequencing variable regions of the genome such as the ribosomal Internal Transcribed Spacer region (ITS), Inter-Genic Spacer region (IGS), Large Sub-Unit (LSU) and Small Sub-Unit rRNA (SSU rRNA) gene (Huang et al., 2008;Santin et al., 2009;Dong et al., 2010;Li et al., 2012).In fungi, the noncoding spacer regions of rDNA, which evolve rapidly, have been utilized in inferring phylogeny among more closely related taxa.The IGS region have been examined in the course of evolutionary and taxanomic studies of fungi (Erland et al., 1994;Molin et al., 1993;Aminnejad et al., 2009).Nowadays, the noncoding spacer regions (ITS/IGS) have been found valuable to study and establish relationships among closely related taxa particularly in fungi and other organisms.The ITS / IGS sequence regions can develop variations within genera that distinguish them at intra species level.The taxonomic value of ITS/IGS region is due to their significant heterogeneity in length and nucleotide sequences.This study targeted the SSU-rRNA gene and IGS region located in between the SSU-rRNA and 5S-rRNA gene cluster of the microsporidia.The IGS region that has the most rapidly evolving sequence provides significant data considered phylogenetically useful for delineating relationships within species (Hillis et al., 1991).
The aim of our study was to identify and delineate microsporidian isolates based on IGS and SSU rRNA gene sequences.In order to analyze the IGS and SSU rRNA gene sequences, we had amplified IGS and SSU rRNA gene followed by cloning and sequencing.We defined and analyzed inter and intra-individual variations in the IGS and SSU rRNA gene sequences of test microsporidian isolates.The heterogeneity of r-DNA sequence derives the genetic variations of isolates and elucidates phylogenetic relationship among defined microsporidian species/strains.

Collection, isolation and purification of microsporidian spores
Two strains of microsporidia were extracted from individual infected moths of A. mylitta collected from the forests areas of Deoghar district Jharkhand, India by maceration and suspended in 0.85% NaCl followed by filtration through layers of cheese cloth and centrifugation at 3500 r/min for 10 min.The spore pellet obtained was further purified through density gradient ultracentrifugation using Percoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) described by Undeen (Undeen and Alger, 1971).The details of microsporidian isolates, places of collection, host and size are given in Table 1.

Spore morphology (length and width)
The morphology of purified fresh spores was observed under phase contrast microscope (Carl Zeiss-AXIO, Humburg, Germany) and measurements were recorded according to the method of Undeen (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.All the measurements are presented in micrometers as mean values of 12 individual observations.

DNA extraction and purification
Genomic DNA was extracted from the sporoplasm using the glass bead method (Undeen and Cockburn, 1989).DNA concentration

Cloning and sequencing of SSU-rRNA gene and IGS region
The purified DNA fragments were ligated into a pJET1.2cloning vector in the presence of T4 DNA ligase (CloneJET PCR Cloning Kit, Thermo Scientific) at 22°C.The ligated products were transfected into JM101 competent cells and plated on ampicillin agar plates.White colonies were selected, and plasmids were isolated from the cells using the GeneJET plasmid miniprep kit (Fermentas Life Sciences).After isolating plasmid DNA from transformed Escherichia coli cells, the plasmids were digested with restriction enzyme (Bgl II) to check whether they contained the desired insert.Three clones from each of the microsporidian isolates were sequenced using DNA sequencing kit (BDT version 3.1) on a semi-automatic DNA sequencer (ABI Prism 310, Applied Biosystems, Perkin Elmer) with M13 universal primers at Eurofins Genomics India Pvt. Ltd., Bangalore, India.The sequences were cleaned of any vector contamination using a vector screen program, (NCBI, Bethesda, Maryland, USA).The final sequences were deposited in NCBI GeneBank and the details are given in Table 2.

Phylogenetic analysis of SSU-rRNA and IGS-Sequences
The analysis of SSU-rRNA and IGS sequence homology was carried out using BLAST search from NCBI database.In contrast, 16 non-redundant microsporidian sequences from Nosema/Vairimorpha species including an out-group Glugoides intestinalis for IGS and Encepalatazoon hellem for SSU-rRNA were retrieved from the NCBI database.These sequences were aligned with IGS and SSU-rRNA gene sequences of test microsporidian isolates including an Indian Nosema reference strain in CLUSTAL W program (Higgins et al., 1994) (Table 3).The molecular phylogenetic trees were constructed from aligned sequences using maximum likelihood using the branch and bound option (with 500 bootstrap replicates) of the MEGA program (Version 6.0) (Tamura et al., 2013).The cloned and sequenced IGS and SSU-rRNA gene sequences of the test isolates and 16 Nosema reference strains were checked for sequence similarity using the Sequence Identity Matrix in BioEdit software (Hall, 1999) (Tables 4 and 5).

Morphological characterization
The spore sizes of both the isolates ranged from 4.50 to 5.10 <mu>m in length and 2.56 to 3.20 <mu>m in width.Details are given in Table 1.

PCR amplification, cloning and sequencing of IGS fragment and SSU-rRNA gene
The PCR amplification for both the microsporidian isolates using targeted IGS primer sets was successfully carried out.The amplified fragment consisted of a partial region of SSU-rRNA gene followed by complete sequences of IGS and 5Sr RNA gene with an expected fragment size of approximately 500 bp Substantial fulllength sequences of the IGS cloned gene was obtained  2.

Length and sequence variation in IGS and SSU-rRNA gene sequences
The IGS and SSU-rRNA gene sequences of 16 different microsporidians species with similar homology downloaded from NCBI were utilized for analysis.The sequence similarity of IGS sequence between test microsporidian isolates was observed to be 86%, while, SSU-rRNA gene sequence similarity was 99%.The average sequence similarity between test isolates and all reference Nosema sp. for IGS was 75%, whereas, 97% similarity was found in case of SSU-rRNA gene.Highest (90%) IGS sequence similarity was observed between the test isolates [MIJ-2pD and MIJ-4cD], and a reference strain Nosema antheraeae, while, both test isolates had least (34%) similarity with Varimorpha reference species (Table 4).On the other hand, SSU-rRNA gene sequence similarity between test and reference microsporidians strains were very high with both test isolates showing about 99% similarity with almost all reference Nosema strains except NIK-1s_mys.Accordingly, it was clearly observed that test isolates had substantial low similarity for IGS sequences compared to SSU-rRNA gene sequences (Tables 4 to 5).As expected, a very low level of similarity was observed between both IGS and SSU-rRNA gene sequences of test isolates and out-group species: Glugoides intestinalis for IGS and Encepalatazoon hellem for SSU-rRNA (Tables 4 to 5).

Phylogenetic analysis based on IGS and SSU-rRNA gene sequences
Two dendrograms were constructed based on IGS or SSU-rRNA gene sequences.In both cases, test isolates separated from reference microsporidia species with above 50% bootstrap value (Figures 1 and 2).The analysis of both phylogenetic trees manifested that the test isolates and 16 Nosema reference strains including an out-group separated into two major clades.In the IGS phylogenetic tree, the test microsporidian isolates and three Nosema reference species [N.philosamia (FJ767862.1),N. antheraeae (DQ073396.1)and N. disstriae (HQ457431)] grouped in a single clade, while in SSU-rRNA gene phylogenetic tree one additional uncultured Nosema sp.(EU338534) joined the test isolate group.The other eleven Nosema reference species including Indian Nosema sp.(NIK-1s_mys)    1 and 2).
In addition, the reference Vairimorpha species separated alone in the same clade with all reference Nosema sp. and test isolates in both generated dendrograms.As expected, G. intestinalis (AF394525) and E. hellem (L19070) used as an out-group in IGS and SSU-rRNA based dendrograms, respectively got separated from both major clades in the constructed trees (Figures 1 and 2).

DISCUSSION
The present study aimed at characterization of two strains of microsporidia isolated from the tasar silkworm A. mylitta based on morphology and phylogenetic analysis of variations in IGS and SSU-rRNA gene sequences.

Morphological characterization
Morphological studies did not reveal any major variations in spore width and length of the isolates.Since spore size for a given species may vary with respect to the host, the morphological characteristics are not considered for delineating identity of microsporidian isolates (Brooks et al., 1972).

Molecular characterization
Genotyping studies based on the ribosomal RNA small subunit gene (SSU-rRNA) sequence analysis are a promising tool for organism identification (Zhu et al., 2010;Ku et al., 2007).
The ribosomal DNA comprises of highly conserved SSU gene sequences that occur in multiple copies within the eukaryotic genome and has been widely exploited since many years, for deriving phylogeny inference (Liu et al., 2012).The phylogenetic relationships of several micro-sporidian genera including Vairimorpha and Nosema were studied based on their SSU-rDNA sequence similarity (Baker et al., 1994;Dong et al., 2010).Three different microsporidia species showed 100% identity of SSU r-RNA gene sequences while their ITS, IGS and 5S sequences varied (Dong et al., 2010).Molecular variations have been established among Trichosporon isolates from close geographical locations based based on restriction patterns of IGS (Sugita et al., 2002).
Various studies have indicated that, molecular phylogenetic analysis based on IGS region might be a better tool to investigate intra-specific divergence and would provide significant molecular evidence for classification and evolutionary studies of microsporidia (Dong et el., 2010;Sagastume et al., 2010;Liu et al., 2013).A recent report indicated differentiating of N. ceranae strains from different geographic origins in Europe based on sequence analysis of highly variable regions of IGS sequence and a part of r-RNA that corresponded to IGS region and their virulence (Dussaubat et al., 2012).
Based on these reports we targeted the IGS as well as SSU-rRNA sequences for phylogenetic analysis of key microsporidia isolates and also made a comparative study of the said gene sequences with 16 different Nosema/Vairimorpha strains to derive a phylogenetic inference for both microsporidian strains isolated from A. myllita.The IGS and SSU-rRNA gene sequences of the isolates indicated that they belong to the genus Nosema.
Comparative sequence similarity analysis of IGS and SSU-rRNA sequence for the test isolates and reference strains revealed that SSU-RNA gene sequence is highly conserved.Sequence similarity analysis revealed that, the IGS sequence (280 bp) showed 70% similarity between test and reference microsporidian isolates while SSU-rRNA (1240 bp) showed 99% similarity.The dendrograms of both test isolates based on IGS and SSU-rRNA gene sequences revealed close genetic relationship with three Nosema species, that is, Nosema philosamia, and Nosema antheraeae from China and Nosema disstriae from Canada.Since, the test isolates were extracted from Antheraea mylitta the genetic similarity of test isolates with N. philosamia, and N. antheraeae support host specification of the microsporidian strains.The trees generated from the IGS and SSU-rRNA gene sequences of the test microsporidia isolates revealed a closed as well as complex phylogenetic relationship of the test isolates with each other due to evolutionary process.Thus, the sequence based phylogenetic analysis of IGS and SSU-rRNA genes provided additional molecular evidence for the classification and evolutionary study of microsporidian isolates on species as well as genus level.
In recent times, the ITS/IGS genes of ribosome were utilized for identification of parasite species and strains as well as a tool for molecular diagnosis.The SSU rRNA gene sequence variation used for establishing phylogenetic relationship between different species in the same genus can also be used for the classification of microsporidia at the species level.Consequently, sequence and phylogenetic analysis based on the combination of SSU rRNA gene and IGS of ribosome might provide additional molecular evidence for the classification and study of evolution of microsporidia.Thus, the gives strong evidence of genetic recombination in N. antheraeae or N. philosamia and a molecular method for defining species in closely related Nosema species.Further, it also revealed, the SSU-rRNA and IGS sequence variability among the microsporidian isolates that would be a rich source of information and could serve to differentiate these isolates in order to give us insight into their origins as well as the spread of this

Figure 1 .
Figure 1.Phylogenetic tree based on IGS sequences.Eighteen different microsporidians IGS sequences were analyszed based on maximum likelihood approach using MEGA 6 (Tamura, 2013) run with 500 bootstrap replication.Number of each node indicates bootstrap value.

Figure 2 .
Figure 2. Phylogenetic tree based on SSU-rRNA gene sequences.Eighteen different microsporidians IGS sequences were analyszed based on maximum likelihood approach using MEGA 6 (Tamura, 2013) run with 500 bootstrap replication.Number of each node indicates bootstrap value.

Table 1 .
Details of the microsporidian isolates: Their place of collection, host and morphology.

Table 2 .
Detail of SSU-rDNAs sequences submitted in NCBI of two microsporidian isolates.

Table 3 .
Taxon, source, host and Gene Bank Acc.No. of microsporidian species used in the phylogenetic analysis.

Table 4 .
IGS sequences similarity matrix of 18 different microsporidians strain including two experimental isolates.

Table 5 .
SSU-rRNA gene sequences similarity matrix of 18 different microsporidians strain including two experimental isolates.