ALBERT

Description: 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 incorporation of these particles into the sponge cells. Fluorescence-activated cell sorting (FACS) and fluorescent microscopy were used to quantify and verify phagocytic activity, defined here as the population of sponge cells with incorporated particles. Sponges were incubated with a green microalgae to test if particle concentration in the seawater affects 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., fluorescently-labeled bacteria and fluorescent beads). The percentage of sponge cells that had incorporated algae, bacteria, and beads ranged between 5 to 24 %. These phagocytic sponge cells exhibited different morphologies and sizes depending on the type of particle presented to the sponge. Particle incorporation into sponge cells was positively related to algal concentration in the seawater, 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. 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.

Description: Sponges (Porifera) are basal metazoans that feed on and establish symbiotic associations with microbes. How sponges discriminate between bacteria to digest as food, incorporate as symbionts, or eliminate as pathogens remains unknown. This thesis aimed to study mechanisms for microbial discrimination in sponges. Mechanisms likely to modulate sponge-microbe interactions are introduced (Chapter 1). The host transcriptomic response upon seawater and symbiont microbial consortia encounter was characterized by RNASeq in two sponges (Chapter 2). Aplysina aerophoba showed little differential gene expression and no participation of receptors, whereas Dysidea avara responded by regulating NLRs suggesting that microbial discrimination is driven by the repertoire of immune genes as well as to what degree they are induced. On the cellular level, an in-vivo phagocytosis assay was established in Halichondria panicea combining incubations with microalgae, bacteria, and latex beads with cell dissociation and fluorescence-activated cell sorting (FACS) to quantify particle incorporation into sponge cells (Chapter 3). After 30 min particles were predominantly incorporated into choanocyte-like cells and appeared to be translocated to archaeocyte-like cells after 60 min. Lastly, the established assay was combined with proteomic analysis to investigate H. panicea’s phagocytic response upon exposure to a “native” and a “foreign” Vibrio isolate (Chapter 4). Vibrio incorporation into sponge cells was indiscriminate, but the distribution of vibrios into different cell types differed between isolates. Phagocytic-related proteins were in a higher abundance in the foreign vs. the native treatment. These results indicate that bacterial discrimination in H. panicea occurs after internalization leading to differences in the processing of foreign vs native vibrio types.