Genetic structuring and estimation of reproductive adults in Onchocerca volvulus: A genome-wide analysis across hosts and regions

Abstract

Genomic analysis of parasites can deepen our understanding of their transmission, population structure, and important biological characteristics. Onchocerciasis (river blindness), caused by the parasitic nematode Onchocerca volvulus, involves adult worms residing in subcutaneous nodules that produce larval-stage microfilariae (mf), which are routinely detected in the skin for diagnosis. Whole-genome studies of mf are limited; most analyses have focused on the mitochondrial genome. We conducted a genome-wide analysis with 94% median nuclear genome coverage, analyzing 171, 37, and 98 mf from 16, 3, and 5 individuals from Ghana, Liberia, and the Democratic Republic of Congo, respectively. These data were used to investigate population differentiation, estimate the number of reproductive adult worms, and analyze genetic variation across chromosomes. Population genetic analyses across hosts and countries showed that nuclear genome diversity can reveal fine-scale genetic structure, even between geographically close countries, providing more resolution than mitochondrial haplotype data. By reconstructing maternal and paternal sibships, we estimated the number of reproductively active adult filariae. Comparisons between adult worm estimates from genetic data and nodule observations showed that genetics-based estimates were higher or equal to observed worm counts in 8 out of 9 hosts for female worms and 7 out of 9 hosts for male worms. Our analysis also revealed lower-than-expected X chromosome diversity, consistent with neo-X chromosome fusions in filarial species. This study represents an important step in using nuclear genome data from mf to support onchocerciasis elimination efforts and in developing genetic tools that could inform mass drug administration programs.

Fig 1. Library composition and genome coverage of Onchocerca volvulus microfilariae samples (n = 315), and the criteria for sample exclusion.

(A) Proportion of reads mapped to O. volvulus (Ov) nuclear and mitochondrial genomes, endosymbiont Wolbachia, and Homo sapiens. (B) Breadth of coverage for each of the four genomes at a minimum depth of 10X. (C) Exclusion of samples with a high rate of missing genotype calls (>11%). (D) Exclusion of samples with highly negative inbreeding coefficient values (<-0.05), indicative of possible sample contamination. Cutoffs for exclusion (panels C and D) are represented by dashed lines.

Fig 2. Genetic differentiation of Onchocerca volvulus microfilariae.

(A) Discriminant Analysis of Principal Components (DAPC) based on the countries of origin and the proportions of successful reassignment using 206 mitochondrial genetic variants. (B) DAPC based on the hosts and the proportions of successful reassignment using 206 mitochondrial genetic variants. (C) DAPC based on the countries of origin and the proportions of successful reassignment using 9,227 nuclear genetic variants. (D) DAPC based on the hosts and the proportions of successful reassignment using 9,227 nuclear genetic variants.

Fig 3. Comparison of Onchocerca volvulus adult worm estimates based on nodulectomy and genetic analysis of microfilariae (mf) for participants from Ghana.

Histological evaluation of worm-sections stained with anti-aspartic protease sera (APR) for viability. (A) Adult female with normal embryogenesis. Stretched mf can be seen in uterus. Ut, uterus; i, intestine. (B) Adult female with degenerated embryos. Mf in nodule tissue (arrow). (C) Dead adult female worm. The number of adult worms estimated using mf genetic data and histological analysis of nodules: (D) Adult females, (E) Adult males.

Fig 4. Genome-wide genetic diversity, linkage disequilibrium (LD), and haplotype blocks in Onchocerca volvulus.

Chr 1 (OM1), 2 (OM3), 3 (OM4), and X (OM2) were analyzed using Ghanaian female microfilariae (n = 28). (A) Nucleotide diversity (π) in 500 kb sliding windows. (B) LD decay pattern. Vertical dotted lines indicate the distance at which r² is 0.5 for each chromosome. (C) Box plot of haplotype block length distribution (>10 kb). (D) Proportion of chromosomes found within haplotype blocks. Horizontal dotted lines indicate the percentage of chromosomes found within ≥50 kb haplotype blocks.