Avian malaria, caused by Plasmodium parasites, poses a significant threat to bird populations worldwide, particularly in vulnerable island ecosystems. Yet, progress in understanding avian malaria transmission dynamics has been hampered by the lack of molecular tools to quantify and sex the transmissible stages of the parasite: the male and female gametocytes. This challenge is compounded by the nucleated erythrocytes of avian hosts and the absence of an in vitro culture system, which have historically hindered the advancement of molecular approaches. Here, we develop and validate the first molecular markers to discriminate between asexual, male, and female gametocytes in the widespread avian malaria parasite Plasmodium relictum. Using single-cell RNA sequencing and orthology-guided stage mapping, we identified conserved, stage-specific transcripts and leveraged this information to develop molecular markers capable of quantifying and distinguishing male and female gametocytes in two parasite cyt-b lineages, pSGS1 and pDELURB. These markers outperformed microscopy in sensitivity, detected gametocytes earlier and for longer, and revealed consistently higher male-to-female gametocyte ratios than estimated by blood smears. The high degree of conservation of these markers across Plasmodium species suggests broad applicability of these markers to other avian lineages.
