Microbial interactions were key in the origin of eukaryotes and may have been involved in major eukaryotic transitions to multicellularity, such as the origin of animals. Today, microbial interactions can influence animal multicellular development and are important for immune and digestive system functioning. The closest unicellular relatives of animals have temporary multicellular lifestages and in one lineage, the choanoflagellates, these are triggered by interactions with bacteria. It is thus suggested that microbial interactions were involved in the evolution of the multicellular animals from unicellular ancestors. However, we are currently missing the information needed to determine whether this was the case. Apart from case studies in choanoflagellates, we have no information about microbial interactions in the closest unicellular relatives of animals, a group comprised of four genetically, morphologically and ecologically diverse lineages. Inferences about the unicellular ancestors of animals are dependent on comparative data from a wide pool of extant organisms, which is currently missing. In this project I will address this critical knowledge gap through a systematic analysis of microbial interactions across unicellular animal relative diversity. The project will entail the following research plans. Using in-situ cultivation and single-cell metagenomics I will identify which prokaryotic groups participate in interactions with diverse unicellular animal relatives. I will then investigate the effects of interaction with select prokaryotes on unicellular animal relative multicellular behaviour and gene transcription. With this information, I will then investigate how genes mediating interactions evolved across the unicellular-to-animal transition and whether there were any horizontal gene transfers from potential prokaryotic interaction partners. With the results I can then reconstruct contributions from microbial interactions on the path from unicellular ancestor to animal. Ultimately, through this project we hope to determine whether, and if so how, microbial interactions were involved in the origin of animals and gain insight into multicellular evolutionary transitions.