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Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates’ closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied.

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1.AbstractTunicates are highly diverse marine invertebrate filter-feeders that are vertebrates’ closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually through budding, typically leading to the formation of colonies where animals, called zooids, are interconnected by an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.We here present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km from their natural habitat. We show that colonies adapt well to in-lab culturing and that a specific strain can be isolated, propagated and used for research efficiently over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.HighlightsFirst in-land recirculating aquaculture for colonial ascidiansOver 3 years of continuous breedingSemi-automated setup with minimized maintenanceGood biomass production for strain propagation4 different culture conditions for optimized breeding for species of interest