New mitochondrial genome sequence of Plasmodium pitheci from Bornean orangutans
The mitochondrial (mt) genome of Plasmodium pitheci, the malaria parasite morphologically identified in Bornean orangutans (Pongo pygmaeus), has now been sequenced and analyzed, providing a crucial genetic reference for this Asian NHP malaria lineage. This study fills a long-standing gap in molecular characterization, moving beyond morphology to confirm species identity and to explore evolutionary relationships with other Plasmodium lineages that infect primates.
What was sequenced and how the genome was obtained
Researchers collected blood samples from P. pitheci infections in orangutans managed at rehabilitation centers in Indonesian Borneo and neighboring regions. Genomic DNA was extracted from malaria-infected blood and sequenced using Oxford Nanopore Technologies long-read sequencing. Initial attempts to amplify the complete mt genome from individual infections were challenging, so a targeted, primer-based approach was employed to amplify roughly 5.8 kb of the mitochondrial genome across 23 samples. These reads were mapped to reference Plasmodium mt genomes, and a consensus sequence was generated for the final assembly. The mt genome consists of the standard Plasmodium-typical content, including three protein-coding genes and rRNA components, mirroring the conserved mitochondrial structure observed in other malaria parasites.
Genomic features and conservation of the Plasmodium mt genome
The completed P. pitheci mitochondrial genome spans about 5.87 kb with a GC content around 35%. It exhibits the conserved organization seen across Plasmodium species, encoding Cytochrome c oxidase subunits and cytochrome b, along with the small and large ribosomal RNA subunits. This structural conservation underscores the essential role of the mitochondrial electron transport chain in the parasite’s life cycle and highlights why mtDNA is a reliable resource for phylogenetic and population studies in Plasmodium.
Phylogenetic placement and lineage connections
Bayesian phylogenetic analyses using whole mt genomes and mt-COI sequences show that P. pitheci forms a monophyletic group with Plasmodium sp. VM, VS, and Pongo clade A/B lineages reported from orangutans. The clustering supports the idea that these lineages share a recent common ancestor and may represent geographic variants of a single species rather than distinct, fully separate species. A closely related P. sp. Pongo clade C, however, diverges more, with genetic distances similar to those observed between established species such as P. inui and P. hylobati. These findings suggest a nuanced evolutionary history for orangutan-associated malaria parasites in Southeast Asia, including potential ongoing diversification or geographic speciation.
Haplotype analysis across 48 mtDNA sequences, combining new data with GenBank entries, revealed 27 distinct haplotypes. West Kalimantan samples dominated by a single haplotype indicate a possible founder effect or localized transmission, while Central Kalimantan and Sabah displayed greater diversity, hinting at more complex population structure. Tajima’s neutrality test showed a negative value, consistent with population expansion or purifying selection, consistent with recent dynamics in the parasite population tied to host movements and conservation-related translocations.
Implications for orangutan conservation and malaria management
Understanding the mt genome of P. pitheci provides a scalable tool for species-specific detection, improving diagnosis and treatment of malaria in Bornean orangutans. For conservation programs, the data offer a baseline to monitor parasite diversity as orangutans are moved between sites or released back into the wild. The study also highlights the zoonotic caution surrounding NHP malaria: while P. pitheci is currently associated with orangutans, Southeast Asia’s overlapping distributions of NHP and human malaria parasites necessitate surveillance to preempt potential spillover or cross-species infections, particularly as human–wildlife interfaces increase with habitat change and rehabilitation efforts.
Future directions
Further sequencing of additional P. pitheci isolates from diverse geographic regions would illuminate population structure and historical demography. Whole-genome comparisons with other Plasmodium species, including those infecting humans and other primates, could elucidate evolutionary pressures shaping parasite diversity in Asia and help refine diagnostic probes for species-specific detection in field and clinical settings.
Conclusion
The successful assembly and annotation of the Plasmodium pitheci mitochondrial genome from Bornean orangutans marks a significant step in primate malaria research. By clarifying evolutionary relationships and revealing haplotype diversity, this work supports targeted conservation strategies and underscores the need for continued molecular surveillance of malaria parasites at the human–NHP interface in Southeast Asia.
