All raw data accompanying the manuscript.

Hammerhead ribozymes have found extensive applications in gene expression regulation across diverse biological systems including Escherichia coli, yeast, plants, and mammalian cells. However, their implementation in parasitic nematodes remains unexplored. Strongyloides stercoralis emerges as a particularly valuable model organism for studying developmental transitions in parasitic nematodes due to its unique life cycle alternating between parasitic and free-living stages. To expand the experimental toolkit for investigating developmental, evolutionary, and behavioral processes in this species, we established a conditional gene regulation system through transgenic integration of synthetic ribozyme constructs and demonstrated efficacy in regulating both exogenous (mrfp) and endogenous (unc-22) gene expression through targeted RNA processing mechanisms. Focusing on the insulin/IGF-1 signaling pathway, a critical regulator of parasitic nematode development and longevity, we implemented ribozyme-mediated post-transcriptional control to dissect functional divergence between two isoforms of the insulin receptor homolog Ss-DAF-2. Comparative analysis revealed isoform-specific characteristics: while both isoforms maintain conserved signaling functions, isoform B exhibits specific binding affinity for human insulin and demonstrates significant transcriptional upregulation during parasitic transition phases. This ligand selectivity profile suggests that isoform B may serve as a molecular interface for host-derived insulin signaling coordination during parasitism. This study established a programmable ribozyme tool in S. stercoralis, functionally discriminated the two Ss-DAF-2 isoforms through precision RNA engineering, and identified isoform-specific ligand preferences with implications for host-parasite signaling. Our findings not only validate ribozyme-based approaches for genetic manipulation in parasitic nematodes but also lay the groundwork for future implementation of synthetic RNA switches in helminth research.