Connecting structure and function from organisms to molecules in small animal symbioses through chemo-histo-tomography

Abstract

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their body is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histological changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. To close this gap, we developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines spatial metabolomics based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (microCT) on the same animal. Both high-resolution MSI and microCT allowed us to correlate the distribution of metabolites to the same animals three-dimensional (3D) histology down to sub-micrometer resolutions. Our protocol is compatible with tissue specific DNA sequencing and fluorescence in situ hybridization (FISH) for the taxonomic identification and localization of the associated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an interactive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and microCT, we introduce a workflow to connect metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem-functioning. SignificanceMetabolites mediate the establishment and persistence of most inter-kingdom symbioses. Still, to pinpoint the metabolites each partner displays upon interaction remains the biggest challenge in studying multi-organismal assemblages. Addressing this challenge, we developed a correlative imaging workflow to connect the in situ production of metabolites with the organ-scale and cellular 3D distributions of mutualistic and pathogenic (micro)organisms in the same host animal. Combining mass spectrometry imaging and micro-computed X-ray tomography provided a culture-independent approach, which is essential to include the full spectrum of naturally occurring interactions. To introduce the potential of combining high-resolution tomography with metabolite imaging, we resolve the metabolic interactions between an invertebrate host, its symbiotic bacteria and tissue parasites at unprecedented detail for model and non-model symbioses.