Co-transmission of Related Malaria Parasite Lineages Shapes Within-Host Parasite Diversity

Abstract

In high-transmission regions, we expect parasite lineages within complex malaria infections to be unrelated due to parasite inoculations from different mosquitoes. This project was designed to test this prediction. We generated 485 single-cell genome sequences from fifteen P. falciparum malaria patients from Chikhwawa, Malawi-an area of intense transmission. Patients harbored up to seventeen unique parasite lineages. Surprisingly, parasite lineages within infections tend to be closely related, suggesting that superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Both closely and distantly related parasites comprise an infection, suggesting sequential transmission of complex infections between multiple hosts. We identified tetrads and reconstructed parental haplotypes, which revealed the inbred ancestry of infections and non-Mendelian inheritance. Our analysis suggests strong barriers to secondary infection and outbreeding amongst malaria parasites from a high transmission setting, providing unexpected insights into the biology and transmission of malaria.

Keywords: genetics; genomics; malaria; population structure; single cell sequencing; transmission.

Figure 1.. The within host genetic diversity of malaria parasites is shaped by mosquito transmission.

(A) A simple monoinfection is generated when an uninfected individual is bitten by a mosquito bearing a single parasite genotype. (B) A superinfection occurs when an individual is bitten by two mosquitoes, each bearing a single parasite genotype. (C) Co-transmission of parasites occurs when a single mosquito bearing multiple genetically distinct parasites bites an uninfected individual. As genetic recombination is an obligate stage of mosquito transmission multiple related parasites may infect an individual through this route.

Figure 2.. Complexity of infection inferred from bulk and single-cell sequencing.

(A) F_WS scores for 49 bulk sequenced infections. Infections above the dashed line (FWS=0.95) are assumed to be clonal. (B) Inferred number of haplotypes (K) inferred by DEploid, infections are ordered by the F_WS score. Black dots in (A) and (B) denote infections also deconvoluted by single-cell sequencing. (C) Number of unique haplotypes inferred by single-cell sequencing. This data is included in Table S1.

Figure 3.. Inferring the composition of malaria infections from bulk sequencing data.

Correlation between the effective number of haplotypes inferred from single-cell sequencing and either F_WS (A) or Effective K inferred from DEploid (B). (C) The maximum similarity between each haplotype inferred by DEploid and single-cell sequences from the same infection. The abundance of the haplotype (as estimated by DEploid) for each haplotype is shown on the y- axis. Majority haplotypes were inferred with high accuracy (73.6–99.9% similarity), with reduced accuracy for minority haplotypes. After exclusion of low abundance (<10%) haplotypes and haplotypes from clonal infections there was a significant relationship between abundance and maximum similarity (adjusted r²=0.32, p=4.6×10⁻⁵, linear model). Clonal infections (MAL29, MAL31, MAL38) and simple superinfections (MAL5) perform uniformly well with DEploid, though other infections show variable success.

Figure 4.. A network representation of pairwise IBD sharing across the genomes.

Each node represents a single parasite colored by the infection of origin. Nodes are joined if >15% of the genomes are shared IBD. Each node is colored by the infection it was derived from, with bulk sequences denoted by a square and single-cell sequences by a circle. The parasite from a single infection are highlighted by dashed lines. MAL23 and MAL48 both contain multiple unlinked clusters indicative of superinfected parasites.

Figure 5.. Recent ancestry inferred from IBD sharing.

(A) Density plot of the total IBD shared between parasites from a single infection (labeled to the left of the plot). (B) Density plot of the mean IBD block length between parasites from a single infection. The dotted lines in A and B show the expected value for parasites separated by differing number of meiosis, e.g. clonally identical (~21Mb total IBD/~1.5Mb Mean IBD length) and separated by a single meiosis (~10Mb total IBD/~0.6Mb Mean IBD length). The most distant relationship shown is 5 meiosis. (C) Relative frequency of different degrees of relatedness inferred between parasites from the same infection using the ERSA algorithm (ns - no significant relatedness observed). (D) The number of mitochondrial haplotypes identified in this infection.

Figure 6.. Patterns of recombination in a tetrad formed during meiosis.

Two parental haplotypes (red and blue) were inferred using Hapi and the inheritance of these haplotypes was inferred in the four parasite genotypes shown here. Across the genome there is consistent 1:1 inheritance of parental genomes aside from the proximal end of chromosome 4 and the distal end of chromosome 2 (green boxes, also shown expanded). Inbreeding among the ancestors of the parental genotypes has eroded variation on chromosomes 8 and 9 (orange boxes).