A novel in-vivo phagocytosis assay to gain cellular insights on sponge-microbe interactions

Sponges harbor diverse, specific, and stable microbial communities, but at the same time, they efficiently feed on microbes from the surrounding water column. This filter-feeding lifestyle poses the need to distinguish between three categories of bacteria: food to digest, symbionts to incorporate, and pathogens to eliminate. How sponges discriminate between these categories is still largely unknown. Phagocytosis is conceivable as the cellular mechanism taking part in such discrimination, but experimental evidence is missing. We developed a quantitative in-vivo phagocytosis assay using an emerging experimental model, the sponge Halichondria panicea. We incubated whole sponge individuals with different particles, recovered the sponge (host) cells, and tracked the particles into the sponge cells to quantify the sponges phagocytic activity. Fluorescence-activated cell sorting (FACS) and fluorescent microscopy were used to quantify and verify phagocytic activity (i.e., the population of sponge cells with internalized particles). Sponges were incubated with a green microalgae to test the effect of particle concentration on the percentage of phagocytic activity, and to determine the timing where the maximum of phagocytic cells are captured in a pulse-chase experiment. Lastly, we investigated the application of our phagocytic assay with other particle types (i.e., bacteria and fluorescent beads). The percentage of phagocytic cells that had incorporated algae, bacteria, and beads ranged between 5 to 24 %. We observed that the population of phagocytic sponge cell exhibited different morphologies and sizes depending on the type of particle presented to the sponge. Phagocytosis was positively related to algal concentration suggesting that sponge cells adjust their phagocytic activity depending on the number of particles they encounter. Our results further revealed that sponge phagocytosis initiates within minutes after exposure to the particles. Fluorescent and TEM microscopy rectified algal internalization and potential digestion in sponge cells, and suggests translocation between choanocyte and archeocyte-like cells over time. To our knowledge, this is the first quantitative in-vivo phagocytosis assay established in sponges that could be used to further explore phagocytosis as a cellular mechanism for sponges to differentiate between different microorganisms.