Seeing the earthworm in a new light

The atlas combines data of mass spectrometry imaging (MSI), fluorescence in situ hybridization (FISH) and microtomography (micro-CT). Credit: Max Planck Institute for Marine Microbiology/PNAS ref.

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Earthworms experience constant chemical interactions with bacteria, fungi, plants, and small invertebrates across soil ecosystems. Even within their tissues, earthworms harbor symbiotic microbes and small animal parasites that trigger internal metabolic responses such as innate immunity. To reveal the fundamental processes that enable animal-microbe symbioses to form and persist, we have to study their metabolic interactions in situ. By combining novel imaging techniques, a team of researchers around Benedikt Geier from the Max Planck Institute for Marine Microbiology (MPIMM) in Bremen, Germany, has now succeeded in imaging the exciting variety of interactions that take place inside the earthworm. This allows us to observe them in a completely new light.

Our understanding of the chemical interactions between small animals and the microorganisms that live inside their bodies is extremely limited. This gap of knowledge has its origin in a methodical challenge: To study the metabolic interactions in symbiosis we have to know who is producing which metabolic product wherein the body. The core of this problem, however, is not only to imagine how molecules are distributed at the micrometer scale. Also, the chemical images are almost impossible to interpret without knowing if a tissue sample is healthy, diseased, or infected with beneficial or pathogenic microbes or even animal parasites.

The new combination of high-resolution imaging techniques can offer a solution to this problem, as the research team now shows.

Geier, the first author of the paper said, in our study, we introduce chemo-histo-tomography, a special three-dimensional imaging approach of the chemistry and anatomy of millimeter-sized animals and their parasites at a cellular level. This method offers a new strategy to visualize the most fundamental processes metabolic interactions in small animal symbioses. It allows us to spatially assign metabolic products to the animal host and its microbial partners at the micrometer scale.

Chemo-histo-tomography (CHEMHIST) combines chemical imaging of metabolites based on MALDI mass spectrometry imaging with the microanatomy of the same animal that is recorded with micro-computed X-ray tomography. The latter is a non-invasive approach allowing X-ray imaging of the 3D histology and can reach a subcellular resolution. For metabolite imaging, MALDI mass spectrometry techniques have become a state-of-the-art tool to visualize micrometer-scale, natural distributions of metabolites. This enables a spatial assignment between chemical profiles and their production site and possibly to their producer.

Manuel Liebeke, leader of the Research Group Metabolic Interactions at the MPIMM and senior author of the study said, this advance allowed us to take an earthworm from the environment and create a 3D atlas of its chemical and physical interactions with the microorganisms naturally occurring inside its tissues. However, we were interested in more than just the biology of the earthworm. Our objective was to also make CHEMHIST applicable to animals directly sampled from their natural habitat, which can be essential for symbiosis research. The resolution of CHEMHIST outperforms existing similar methods developed for medical research in mice by up to two orders of magnitude. This may also lead to new avenues for research on insects or corals, which are key models for symbiosis research, both on land and in water.

Using the combination of different in situ imaging techniques through CHEMHIST, the researchers at the MPIMM in Bremen uncovered metabolites products of the metabolism in the earthworm that could shed light on how it chemically defends itself against parasites and how these, in turn, protect themselves against the earthworm's immune response. However, to even recognize that the chemical image data were metabolites of parasites in earthworm tissue, the 3D anatomical model was indispensable. A high-resolution micro-computer tomograph at the German Electron Synchrotron in Hamburg first made it possible to identify the parasites in the tissue.

Notably, metabolic interactions between animals and their microbes are not restricted to symbiotic tissues. Along the gut-brain axis, microbial metabolites produced in the gut can affect tissues across the host, even reaching the brain. Therefore, extending correlative chemical imaging into 3D approaches can be crucial for capturing the distribution of metabolites involved in symbiotic interactions and thus show how chemical signals from microbes possibly affect crucial processes in their host. Also, the method can be used in many ways: The research team around Liebeke and Geier is currently applying it to deep-sea mussels.

Bernhard Ruthensteiner, leader of the section Invertebrate Varia at the Bavarian State Collection of Zoology said, since I have specialized in the 3D visualization of invertebrate anatomy for years, it was particularly interesting for me to see the molecules hidden behind the morphological structures. The results of the study were made possible by an interdisciplinary collaboration that brought together scientists from the fields of microbiology, zoology, chemistry, and physics. It quickly became clear that visualization such as the correlative 3D atlas of the worm facilitates scientific communication of the data.

Journal information: Benedikt Geier et al, Connecting structure and function from organisms to molecules in small-animal symbioses through chemo-histo-tomography, Proceedings of the National Academy of Sciences (2021). DOI: 10.1073/pnas.2023773118