Background: Orang-utan malaria and the Plasmodium pitheci discovery
Malaria in non-human primates (NHP) spans a diverse genus, with Plasmodium species infecting a broad host range. Among Asian NHPs, orang-utans (Pongo spp.) harbor Plasmodium pitheci and Plasmodium sylvaticum, parasites historically identified by morphology but lacking validated molecular characterization until now. The present work focuses on the complete mitochondrial (mt) genome sequence of P. pitheci from Bornean orang-utans (Pongo pygmaeus), a critically endangered species whose conservation is impacted by infectious disease dynamics and habitat pressures.
Methods: from blood samples to a complete mitogenome
Researchers sequenced mtDNA from 23 blood samples of morphologically P. pitheci-infected orang-utans sourced from conservation centers in West and Central Kalimantan. A two-stage approach combined long-read sequencing on Oxford Nanopore devices with targeted PCR amplification to recover the ~5.8 kb mitochondrial genome, followed by assembly, polishing, and annotation. The study used stage-wise strategies to ensure accuracy and species-level confirmation, including designing species-specific primers and mapping reads to Plasmodium mt references. Phylogenetic analyses employed Bayesian methods on both whole mt genomes and mt-COI sequences to determine relationships with other orang-utan Plasmodium lineages.
Key findings: a conserved, informative mitochondrial genome
The P. pitheci mitogenome is 5,871 bp with a GC content of 35% and encodes the standard Plasmodium mt suite: cox1, cox3, cytb, plus rRNA components. The genome structure is highly conserved across Plasmodium species, underscoring evolutionary constraints on mitochondrial function through the parasite’s life cycle. Importantly, all assembled mt genomes from different orang-utans showed >99% identity, suggesting a common founder effect or limited geographic diversification within the sampled region.
The mt-COI region also yielded sequences that mapped to P. pitheci, Plasmodium sp. VM/VS, and Pongo clade A/B, indicating close evolutionary ties among orang-utan malaria lineages. A Bayesian tree placed P. pitheci within a monophyletic group that includes Pongo clade A/B variants, supporting a shared recent ancestor among these lineages and possible ongoing diversification (i.e., a geographic radiation within orang-utans).
Genetic diversity and population signals
A haplotype analysis of 48 mt sequences (23 from this study, 25 from GenBank) identified 27 distinct haplotypes (Hd = 0.925), with West Kalimantan samples dominated by a prevalent haplotype, while Central Kalimantan and Sabah showed greater diversity. Tajima’s D was negative (−0.959), hinting at population expansion or purifying selection; meanwhile, nucleotide diversity was low (π = 0.0032) despite high haplotype diversity. The combination suggests recent divergence from a common ancestor or historical bottlenecks followed by expansion, strengthening the case that orang-utan malaria lineages harbor rich local diversity despite limited sequence variation at the nucleotide level.
Phylogenetic and taxonomic implications
Phylogenetic analyses consistently linked P. pitheci with orang-utan associated Plasmodium lineages rather than human malaria parasites. The data reinforce the view that Southeast Asian orang-utans carry distinct Plasmodium lineages, with sp. VM/VS and Pongo clade A/B representing closely related variants. Notably, Plasmodium sp. Pongo clade C showed greater divergence, potentially representing a separate species such as P. sylvaticum or an as-yet-unknown lineage. These results enrich our understanding of Plasmodium evolution and highlight the Asian NHP malaria diversity that may illuminate the origins of human malaria parasites.
Conservation and public health considerations
The study underscores that orang-utans can harbor malaria parasites with zoonotic potential, a concern given increasing human–NHP interfaces. For Bornean orang-utans, malaria infections can be clinically significant and influence health management in both wild populations and rescue centers. The clarified mitogenome provides a foundation for species-specific diagnostics and surveillance, improving treatment outcomes and informing translocation or reintroduction strategies. Continued genomic characterization of Plasmodium in orang-utans will advance conservation efforts by refining our understanding of parasite transmission dynamics and their implications for orang-utan health and habitat management.
Future directions
Whole-genome Plasmodium sequencing, broader geographic sampling, and longitudinal studies across ORCs will help resolve population structure, recombination events, and potential spillover risks. Comparative genomics with human malaria parasites could reveal conserved drug targets or resistance markers while clarifying how Plasmodium diversification in Southeast Asia shapes global malaria evolution.
