An improved Stentor coeruleus genome and time-resolved transcriptomics link cyclic nucleotide-dependent kinase signaling to single-cell habituation

The giant ciliate Stentor coeruleus is a single cell capable of modifying its behavior through experience. When repeatedly disturbed, it undergoes habituation, a simple and widely conserved form of non-associative learning, despite lacking a nervous system. How a single cell achieves such behavioral plasticity at the molecular level remains poorly understood. Here, we report a compact, contiguous macronuclear reference genome for S. coeruleus, substantially improving genome continuity and annotation quality while resolving two successive rounds of whole-genome duplication and extensive lineage-specific gene-family expansion. Anchoring time-resolved transcriptomics to this reference, we profiled pooled RNA from behaviorally tracked cells collected at defined points during a mechanical stimulation paradigm and identified 341 genes with significant temporal expression dynamics during habituation. The dominant transcriptional signature of habituation was the coordinated, sustained up-regulation of a conserved cyclic nucleotide, calcium, and phosphatase signaling module centered on five cGMP-dependent protein kinase (PKG) paralogs together with a voltage-gated calcium channel and a protein phosphatase 2A regulatory subunit, representing conserved intracellular components of the neuronal long-term depression (LTD) pathway. By integrating an improved genome with time-resolved transcriptomics of habituation, this work provides a community resource for Stentor and identifies a conserved signaling program as a candidate molecular framework underlying single-cell habituation.