Single-cell transcriptome atlas of adult male and female human hookworm Ancylostoma ceylanicum.

Hookworms infect over 500 million people globally and are a major neglected tropical disease. Their high prevalence and reinfection rates, combined with emerging drug resistance, highlight the need for new therapeutic or vaccine targets. To advance our fundamental understanding, we present a single-cell transcriptomic atlas of male and female Ancylostoma ceylanicum, providing insights into sex-specific gene expression and cellular diversity. We identified 10 distinct cell types in females and 11 in males by comparing marker genes with tissue-specific annotations. These included non-reproductive tissues (pharynx, intestine, muscle, and hypodermis), cephalic and pharyngeal glands important for parasitism and host-parasitic interactions, and tissues important for reproduction (ovaries, testis, sperm, and seminal vesicle). Anatomical confocal microscopy and RNA fluorescence in situ hybridization visualized and validated tissue-specific markers. Our findings reveal previously undescribed molecular pathways and markers, offering insights into hookworm biology and potential intervention targets. This atlas provides a comprehensive cellular framework for this neglected parasite.