Genome-Wide Analyses of Individual Strongyloides stercoralis (Nematoda: Rhabditoidea) Provide Insights into Population Structure and Reproductive Life Cycles

Abstract

The helminth Strongyloides stercoralis, which is transmitted through soil, infects 30-100 million people worldwide. S. stercoralis reproduces sexually outside the host as well as asexually within the host, which causes a life-long infection. To understand the population structure and transmission patterns of this parasite, we re-sequenced the genomes of 33 individual S. stercoralis nematodes collected in Myanmar (prevalent region) and Japan (non-prevalent region). We utilised a method combining whole genome amplification and next-generation sequencing techniques to detect 298,202 variant positions (0.6% of the genome) compared with the reference genome. Phylogenetic analyses of SNP data revealed an unambiguous geographical separation and sub-populations that correlated with the host geographical origin, particularly for the Myanmar samples. The relatively higher heterozygosity in the genomes of the Japanese samples can possibly be explained by the independent evolution of two haplotypes of diploid genomes through asexual reproduction during the auto-infection cycle, suggesting that analysing heterozygosity is useful and necessary to infer infection history and geographical prevalence.

Fig 1. Variant position percentage/numbers across sequence classes.

(A) Intergenic—variant resides in the intergenic region, not included in upstream or downstream regions. Intronic—variant overlaps an intron. Exonic—variant overlaps a coding region. Upstream—variant overlaps a 1-kb region upstream of the transcription start site. Downstream—variant overlaps a 1-kb region downstream of the transcription termination site. Upstream;downstream—variant is located in both downstream and upstream region (possibly for 2 different genes). Genome percentages of the same classes are shown alongside. (B) Effects of the exonic variants. Nonsynonymous—a single nucleotide change that changes an amino acid residue; Synonymous—a single nucleotide change that does not change an amino acid residue; Frameshift—an insertion or deletion of one or more nucleotides that cause a frameshift; Stop gain/loss—a nonsynonymous SNP or indel that creates or eliminates a stop codon at the variant site; Unknown—unknown function (caused by errors in the gene-structure definition in the database).

Fig 2. Principal component analyses of variant data.

(A) a plot including the reference genome strain (USA). Variances represented by PC1 = 40.1% and PC2 = 14.1%. (B) Japanese and Myanmar samples only. Variance represented by PC1 = 28.4% and PC2 = 10.3%. Totals of 234,398 and 128,040 variant positions were included in the PCA analysis for Fig 2A and 2B, respectively.

Fig 3. Pair-wise genetic distances (π) of genomes between samples.

Distances between samples from Japan and Myanmar (green dots) were generally higher than those of the same country. Inter-host comparisons (red dots) show higher πvalues than those of within-host comparisons (blue dots) in Myanmar. The differences are ambiguous in the Japanese samples.

Fig 4

(A) Phylogenetic network analyses based on SNPs in the genome of S. stercoralis. (B) A maximum likelihood tree of SNPs in the mitochondrial genomes of S. stercoralis samples. The scale bars show the number of nucleotide substitutions per site. Branches marked with \\ indicate a two-fold shortening of branches (only for practical purposes).

Fig 5. Intra-genomic heterozygosity in autosomes and sex chromosomes.

Frequencies of heterozygosity (π_t) per nucleotide site for the autosomal and sex chromosomal scaffolds are plotted in x and y axis, respectively. Three areas were shown by different colours and shapes. Broken line represents Y = X.