Characterization of co-infections of haemosporidian parasites in Swinhoe’s pheasant (Lophura swinhoii): Utilizing nanopore sequencing for species-level detection and mitochondrial-genome analysis

Avian haemosporidian parasites are vector-borne apicomplexans that infect bird species globally and pose considerable challenges in detection due to frequent co-infections and morphological convergence. In the present study, we first used Oxford Nanopore Technologies (ONT) to resolve co-infections of haemosporidians in Swinhoe’s pheasant (Lophura swinhoii), an island-endemic galliform. Blood smears revealed two morphologically distinct gametocyte forms: roundish and circumnuclear, and molecular analyses identified three mitochondrial lineages: two novel Haemoproteus lineages (hLOPSWI01 and hLOPSWI02) and one Plasmodium lineage (pNILSUN01). Phylogenetic reconstruction of mitogenomes resolved hLOPSWI01 and hLOPSWI02 within the Parahaemoproteus clade, whereas pNILSUN01 clustered in the Giovannolaia-Haemamoeba clade. Overall, this study revealed the efficacy of ONT in resolving cryptic co-infections through unfragmented mitogenome assembly, overcoming ambiguities inherent to Sanger sequencing. Our findings establish baseline haemosporidian diversity in L. swinhoii and highlight the necessity of combining long-read genomics with morphological scrutiny for accurate parasite taxonomy, particularly in understudied avian hosts facing conservation threats.