Approximately 800 million people worldwide are infected with one or more species of skin-penetrating nematodes. These parasites persist in the environment as developmentally arrested third-stage infective larvae (iL3s) that navigate toward host-emitted cues, contact host skin, and penetrate the skin. iL3s then reinitiate development inside the host in response to sensory cues, a process called activation. Here, we investigate how chemosensation drives host seeking and activation in skin-penetrating nematodes. We show that the olfactory preferences of iL3s are categorically different from those of free-living adults, which may restrict host seeking to iL3s. The human-parasitic threadworm
Strongyloides stercoralisand hookworm Ancylostoma ceylanicumhave highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the S. stercoralis tax-4gene abolishes iL3 attraction to a host-emitted odorant and prevents activation. Our results suggest an important role for chemosensation in iL3 host seeking and infectivity and provide insight into the molecular mechanisms that underlie these processes.