Download files
Abstract
Microorganisms are known to dominate the marine environment, where they play a pivotal role in global nutrient cycling. Within this environment, marine bacteria often trade their free-living lifestyle for a symbiotic relationship with a eukaryotic host. Sponges, which are known to form stable associations with diverse microorganisms, are among the oldest of the extant multicellular organisms, thus representing an ideal model to study the basis for the evolution of the bacteria-animal symbiosis. The function of the sponge-associated microorganisms and the mechanisms underlying their symbiotic interactions are, however, still largely unknown.
Fluorescence in situ hybridisation (FISH) has offered valuable insights into the putative functional characteristics of sponge-associated microorganisms, particularly when linked to metabolic predictions. FISH can also be used in combination with cultivation techniques, which provides a way to test ecological, physiological and biochemical hypotheses raised from multi-omics and imaging data. Therefore, the aim of this thesis was to use FISH to localise and isolate sponge-associated and free-living microorganisms to shed light into their putative function in the marine environment.
FISH was first used to investigate the co-localisation of dominant members in the microbiome of the sponges Cymbastela concentrica and Tedania anhelans. The results showed that dominant phylotypes co-localised according to specific patterns that supported metabolic predictions of their contribution to the host’s carbon and nitrogen metabolism.
Subsequently, FISH was employed in combination with high resolution microscopy techniques to provide insights into the localisation and putative functioning of symbiosis factors, in particular proteins with ankyrin (ANK) repeats. The results showed that a specific ANK protein (namely SSA4) interacted with different cell types in different forms in C. concentrica, and this confirmed an evolutionary conserved mechanism of bacteria-eukaryote interaction.
To contribute to the advancements of cultivation techniques, FISH was also coupled to fluorescence-activated cell sorting (FACS). A protocol called live-FISH was developed, which enabled the specific isolation of living bacteria from artificial and natural microbial communities. This method represents a new technique to isolate and cultivate specific taxa or phylogenetic groups.