Larval settlement is a key step in the life cycles of corals and of many other marine animals, and is also thought to have had general significance in animal nervous system evolution, but its molecular regulation is poorly understood. We will study larval settlement using two complementary cnidarian species: the ecologically relevant scleractinian coral Pocillopora acuta coral and the established hydrozoan experimental model Clytia hemisphaerica. Pocillopora acuta is a promising experimental model pioneer reef-building coral species, with larvae available through monthly emissions in aquarium settings. We will specifically address the hypothesis that one or more specialised cell types at the aboral end of cnidarian larvae (called planulae) express proteins that mediate the settlement process. These include settlement-inducing GLWamide-family neuropeptides that we have already identified in both species. Our central aims are to identify all the participating cells and proteins from the larvae aboral poles, and to characterize the molecular cues from the settlement-inducing biofilms. Exploiting the complementary expertise of the project partners, we will use transcriptomics, peptidomics and metabolomics approaches to identify molecular candidates from the larval (Aim 1) and biofilm (Aim2) sides, followed by functional testing of selected candidate molecules in larval settlement assays (Aim3). For Aim 1 we will exploit our existing Clytia hemisphaerica transcriptome and genome resources, including a single cell transcriptome of the medusa and bulk RNA-seq of the aboral and oral ends of the planula larva. We will additionally produce high-resolution single cell transcriptomes for the Clytia hemisphaerica and Pocillopora acuta larvae with tens of thousands of cells from competent pre-settlement planulae using the 10X Genomics platform, and identify larval cell types using computational pipelines. Cell distributions, morphologies and connections will be determined by in situ hybridisation combined with fluorescence and electron microscopy. We will focus on characterising cell types expressing genes that are candidates for mediating the settlement response, notably small secreted proteins, GPCRs and neuropeptides. In parallel, under Aim 2, we will use mass spectrometry-based approaches to directly identify proteins and small molecules produced during settlement, including antimicrobial type peptides that we speculate may be secondarily detected by the larva and induce settlement. This approach will also allow us to identify biomarkers for larval settlement. The findings from Aims 1 and 2 will together allow us to generate a list of candidate sensory cells, proteins and small molecules that are most likely to initiate the settlement response. Under aim 3 the function of a selection of these will be investigated via the development of larval settlement assays. Established gene knockdown approaches in Clytia will enable us to test the involvement of candidate secreted proteins and their receptors. In both species we will in parallel test the activity of custom-synthesised small molecules to induce the settlement process, and of antibodies to inhibit it. Overall we anticipate that this project will generate a thorough understanding of molecules mediating cnidarian larval settlement. This will contribute to building a picture of larval nervous system evolution, and also potentially have ecological applications.