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Evolutionary Diversity in the Intracellular Microsporidian Parasite Nosema sp. Infecting Wild Silkworm Revealed by IGS Nucleotide Sequence Diversity

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Abstract

Intracellular microsporidian Nosema mylitta infects Indian wild silkworm Antheraea mylitta causing pebrine disease. Genetic structure and phylogeny of N. mylitta are analysed using nucleotide variability in 5S ribosomal DNA and intergenic spacer (IGS) sequence from 20 isolates collected from Southern, Northern and Central regions of Jharkhand State. Nucleotide diversity (π) and genetic differentiation Gst were highest in the Central isolates whereas lowest in the North. Among the isolates, absence of nucleotides, transitions and transversions were observed. Haplotyping showed nucleotide variability at 83 positions in IGS and 13 positions in 5S rDNA. Haplotype-based genetic differentiation was 0.96 to 0.97 whereas nucleotide sequence-based genetic differentiation was higher (Ks = 22.29) between Southern and Central isolates. Bottleneck analysis showed negative value for Tajima’s D and other summary statistics revealing induction of loss of rare alleles and population explosion. From IGS, 17 ancestral sequences were inferred by Network algorithm. Core of nine closely related nodes having ancient nucleotides and peripheral nodes with highly divergent nucleotides were derived. Most diverged peripheral haplotype was Bero (H11) from the Central region whereas Deoghar (H3) of the Northern region diverged early. Phylogeny of N. mylitta grouped Southern and Northern isolates together revealed weak phylogenetic signal for these locations. Phylogeny of N. mylitta with Nosema sp. infecting other lepidopterans clustered N. mylitta isolates with N. antheraea and N. philosamiae of China indicating genetic similarity whereas other species were dissimilar showing diversity irrespective of country of origin.

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Acknowledgements

The authors thank anonymous referees for critical comments that improved the manuscript and Central Silk Board, Bangalore, India, for laboratory facilities. Dr. Wazid Hassan is a recipient of Research Associateship from Council of Scientific and Industrial Research (CSIR), Government of India.

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  1. Molecular Pathology Division, Seribiotech Research Laboratory, Central Silk Board, CSB Campus, Carmelaram Post, Kodathi, Bangalore, Karnataka, 560035, India

    Wazid Hassan & Kangayam M. Ponnuvel

  2. Central Tasar Research and Training Institute, Central Silk Board, Ranchi, Jharkhand, 835303, India

    Basavaraju Surendra Nath

  3. Proteomics Division, Seribiotech Research Laboratory, Central Silk Board, CSB Campus, Carmelaram Post, Kodathi, Bangalore, Karnataka, 560035, India

    Wazid Hassan, Rakesh K. Mishra & Appukuttan Nair R. Pradeep

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Supplementary file1 (TIF 897 kb)

Supplementary Figure 1: (A) Scanning electron microscopy of the N. mylitta spores showing elongated oval shape and smooth surface. Size of the spores ranged from 3.8 - 5.1 µm in length and 2.6 - 3.3 µm in width. (B) PCR profile showed amplification of SSU-IGS-5S rDNA sequence from the genomic DNA extracted from 20 isolates of N. mylitta (1-20). M- Molecular weight marker (Promega)

Supplementary file2 (TIF 1545 kb)

Supplementary Figure 2: Multiple sequence alignment of 5S rDNA sequences from 20 isolates of N. mylitta by Clustal W showing gaps, transitions and transversions. The details of accessions are given in Table 1. * indicates conserved nucleotides.

Supplementary file3 (TIF 4074 kb)

Supplementary Figure 3: Multiple sequence alignment of IGS sequence from 20 isolates of N. mylitta by Clustal W showing gaps, transitions and transversions. The details of accessions are given in Table 1. * indicates conserved nucleotides.

Supplementary file4 (TIF 575 kb)

Supplementary Figure 4: Distribution of informative variable nucleotides at 13 positions in 5S rDNA sequences of 20 isolates of N. mylitta realised after haplotyping using DnaSP software. Ten haplotypes were created out of which haplotypes 3 formed from 11 isolates. Details of haplotypes are given in right column.

Supplementary file5 (TIF 165 kb)

Supplementary Figure 5: Phylogram realised from nucleotide sequences of IGS region of 20 N. mylitta isolates and 20 Nosema species infecting various insects and a microsporidian genus Vairimorpha sp (as an out-group). The sequences were retrieved from the NCBI database (Supplementary Table 1) and aligned using Clustal W program. The unrooted tree was constructed using Neighbor Joining method with branch and bound option and with 1000 bootstrap replicates as given in the MEGA program. All N. mylitta isolates grouped together along with N. antheraea and N. philosamiae. Other Nosema species were grouped into two clusters and Vairimorpha separated as an out group. Details of accessions and host insects are given in Supplementary Table 1.

Supplementary file6 (TIF 147 kb)

Supplementary Figure 6: Phylogram realised from nucleotide sequences of 5S rDNA region of 20 N. mylitta isolates and 20 Nosema species infecting various insects and a microsporidian genus Vairimorpha sp (as an out-group). The sequences were retrieved from the NCBI database (Supplementary Table 1) and aligned using Clustal W program. The unrooted tree was constructed using Neighbor Joining method with branch and bound option and with 1000 bootstrap replicates as given in the MEGA program. All the N. mylitta isolates and other Nosema species grouped together into single cluster and Vairimorpha separated as an outgroup. Details of accessions and host insects are given in Supplementary Table 1.

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Supplementary file8 (DOC 192 kb)

Supplementary file9 (DOC 189 kb)

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Hassan, W., Nath, B.S., Ponnuvel, K.M. et al. Evolutionary Diversity in the Intracellular Microsporidian Parasite Nosema sp. Infecting Wild Silkworm Revealed by IGS Nucleotide Sequence Diversity. J Mol Evol 88, 345–360 (2020). https://doi.org/10.1007/s00239-020-09936-2

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