Chromosome-Contiguous Ancylostoma duodenale Reference Genome from a Single Archived Specimen Elucidates Human Hookworm Biology and Host–Parasite Interactions

Soil-transmitted helminths (STHs) are parasitic nematodes that infect humans, particularly in tropical and subtropical regions, where they contribute substantially to neglected tropical diseases (NTDs). Among them, hookworms (<i>Ancylostoma duodenale</i>, <i>Necator americanus</i> and <i>Ancylostoma ceylanicum</i>) cause substantial morbidity, leading to anaemia, malnutrition, and developmental impairment. Despite the global impact of hookworm disease, genomic research on <i>A. duodenale</i> has lagged behind that of other hookworms, limiting comparative and molecular biological investigations. Here, we report the first chromosome-level reference genome of <i>A. duodenale</i>, assembled from a single adult specimen archived in ethanol at -20 °C for more than 27 years. Using third-generation sequencing (PacBio Revio, Menlo Park, CA, USA, Oxford Nanopore, Oxford, UK), Hi-C scaffolding, and advanced computational tools, we produced a high-quality 319 Mb genome, filling a critical gap in hookworm genomics. Comparative analyses with <i>N. americanus</i> and the related, free-living nematode <i>Caenorhabditis elegans</i> provided new insights into genome organisation, synteny, and specific adaptations. While <i>A. duodenale</i> exhibited strong chromosomal synteny with <i>N. americanus</i>, its limited synteny with <i>C. elegans</i> highlights its distinct parasitic adaptations. We identified 20,015 protein-coding genes, including conserved single-copy orthologues (SCOs) linked to host-pathogen interactions, immune evasion and essential biological processes. The first comprehensive secretome analysis of <i>A. duodenale</i> revealed a diverse repertoire of excretory/secretory (ES) proteins, including immunomodulatory candidates predicted to interact with host structural and immune-related proteins. This study advances hookworm genomics, establishes a basis for the sequencing of archival specimens, and provides fundamental insights into the molecular biology of <i>A. duodenale</i>. The genomic resource for this hookworm species creates new opportunities for diagnostic, therapeutic, and vaccine development within a <i>One Health</i> framework. It complements recent epidemiological work and aligns with the WHO NTD roadmap (2021-2030) and Sustainable Development Goal 3.3.