P-glycoproteins and amphids: a two-stage trajectory of ivermectin resistance in Caenorhabditis elegans.

Ivermectin (IVM) is a cornerstone of nematode control. However, its efficacy is increasingly compromised by emerging resistance in target parasites. P-glycoproteins (Pgps), which mediate drug efflux, and amphid neuron defects, characterized by a dye-filling defective (Dyf) phenotype, may both contribute to IVM resistance in Caenorhabditis elegans, but their respective roles and potential connection remain to be clarified. Here, we investigated these mechanisms in three independently evolved Caenorhabditis elegans lineages exposed to stepwise IVM selection. These populations, although distinct, converged over time to a comparable and stabilized high level of IVM resistance. Endpoint transcriptomes revealed fluctuations in the expression of several pgp, overexpressed in one lineage only, suggesting that sustained pgp upregulation is not a conserved feature of stable resistance. Consistently, neither short-term exposure to IVM nor long-term drug pressure selection elicited strong transcriptional induction. Nevertheless, we uncovered a transient surge of several pgp transcripts during the early selection process of the worms exposed to low doses of IVM. We speculate this relates to an early pgp-dependent tolerance mechanism in the dynamic adaptation program to IVM. Beyond an initial pgp-based response, our expanded RNA-seq analysis across lineages revealed a conserved enrichment for ciliary/neuronal pathways including genes expressed in amphid/phasmid, indicating remodeling of the chemosensory/ciliary network as resistance intensifies. All resistant lines also converged on the Dyf phenotype that emerged during selection at stages coinciding with sharp gains in IVM tolerance, implicating amphid structure/function in the establishment of durable resistance. Together, these data reconcile heterogeneous literature on PGPs by decoupling transient, context-dependent pgp activation from long-term maintenance of resistance, and are consistent with previous work showing ciliary pathways as common denominators of IVM resistance. We propose a two-phase associative model in which early PGP-mediated tolerance precedes the emergence of ciliary/amphidial signatures and a Dyf phenotype during selection that durably reduces susceptibility.