Introduction: Plasmodium pitheci and the orang‑utan malaria puzzle
Malaria parasites in non-human primates (NHP) span over 250 Plasmodium species that infect humans and other primates alike. Among Asian NHPs, orang‑utans (Pongo spp.) host several malaria lineages, including Plasmodium pitheci. Although previous work has described morphological infections and certain genetic markers, a fully validated mitochondrial (mt) genome sequence for P. pitheci from Borneo had not been established until now. This development provides crucial tools for species‑level identification, phylogenetic placement, and understanding how these parasites evolve within orang‑utan populations.
Methods in brief: from blood samples to the complete mt genome
Researchers analyzed 23 blood samples from morphologically confirmed P. pitheci infections in Bornean orang‑utans. Genomic DNA was extracted, and a two‑stage sequencing approach was used. First, long‑read Oxford Nanopore sequencing provided fragmented mt genome data, which was assembled and draft annotated. To obtain a complete, species‑specific mitochondrial genome, researchers designed Plasmodium‑specific primers and performed targeted amplification on all samples, followed by targeted assembly and consensus construction. Annotation leveraged GeSeq with Plasmodium references, and phylogenetic analyses employed Bayesian methods on whole mt genomes and mt‑COI sequences. This rigorous approach confirmed a single, validated P. pitheci mt genome and its relationship to related orang‑utan Plasmodium lineages.
Genome structure and key features
The finalized Plasmodium pitheci mitochondrial genome is 5,871 base pairs long with a GC content of 35%. Its structure mirrors the conserved Plasmodium mt genome, encoding three protein‑coding genes (cox1, cox3, cytb) and the small and large subunit rRNA genes. This conservation underscores the essential role of the mitochondrial electron transport chain in the parasite’s life cycle and explains why mt sequences are highly informative for phylogenetics and species demarcation among malaria parasites.
Phylogeny and lineage relationships: what the mt genome reveals
Bayesian phylogenetic analyses placed P. pitheci in a monophyletic cluster with Plasmodium sp. VM/VS and Pongo clade A/B, underscoring a close evolutionary relationship among orang‑utan Plasmodium lineages. A distinct Plasmodium sp. Pongo clade C shows greater divergence, hinting at a separate species status such as P. sylvaticum or an undescribed lineage. The data support a Southeast Asian reservoir of Plasmodium diversity in orang‑utans and suggest possible geographic structuring or recent speciation among clades A, B, and C. These findings also align with the idea that orang‑utan malarias can be genetically distinct from human malaria parasites, highlighting the deep evolutionary history of Plasmodium in Asian primates.
Genetic diversity and population dynamics
Across 48 mtDNA sequences (23 new P. pitheci genomes and 25 from GenBank), researchers identified 27 haplotypes and an Hd of 0.925, indicating substantial parasite diversity even in a relatively localized host population. Tajima’s D was negative, suggesting an excess of rare variants consistent with population expansion or purifying selection. The combination of high haplotype diversity and low nucleotide diversity mirrors patterns seen in expanding malaria populations, pointing to dynamic transmission histories, possible founder effects, or recent introductions of multiple lineages into orang‑utan rehabilitation centers across Kalimantan and Sabah.
Conservation and public health implications
Understanding the mt genome of P. pitheci has practical benefits for orang‑utan conservation. Species‑specific molecular probes improve diagnosis and treatment in rehabilitation centers, informing strategies that mitigate disease burden in these critically endangered primates. Moreover, the close, yet distinct, relationship of orang‑utan Plasmodium lineages to each other and to other primate malaria parasites enhances our grasp of zoonotic risk. Southeast Asia, as a cradle of diverse Plasmodium lineages, warrants continued surveillance to preempt potential spillovers to humans in areas of close human–NHP contact.
Outlook
The study’s approach—combining long‑read assemblies with targeted amplification—offers a robust template for characterizing Plasmodium mt genomes from other NHP hosts. Expanding sampling across additional locales and host species will sharpen our view of the evolutionary landscape, enable finer population genetics, and support targeted conservation planning for orang‑utans and their malaria parasites.
