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Abstract

Our understanding of metabolic interactions between small symbi-otic animals and bacteria or parasitic eukaryotes that reside within their bodies is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histologi -cal changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. We addressed this challenge and developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines chemical imaging of metabolites based on mass spectrom-etry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (micro-CT) on the same animal. Both high-resolution MSI and micro-CT allowed us to correlate the distribution of metab-olites to the same animal's three-dimensional (3D) histology down to submicrometer resolutions. Our protocol is compatible with tissue-specific DNA sequencing and fluorescence in situ hybridiza-tion for the taxonomic identification and localization of the associ-ated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an in-teractive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and micro-CT, we present a methodological groundwork for connecting metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem functioning.