MALDI-FISH for Colocalization of Brominated Metabolites and Pseudovibrio spp. in Aplysina Tissue

Abstract Bromotyrosine-containing natural products have long been isolated from marine sponges of the order Verongiida . Recent studies have questioned the source of these natural products whether production occurs through the sponge, its diverse microbiota, or perhaps a combination of both. While studying the uptake of engineered bacterial strains by sponges, we have observed production of fistularin-3 in sponge. To explore these results further, we employed multimodal imaging techniques to co-localize microbial metabolites with fluorescent in situ hybridization (FISH) probes. Here, we built on previously developed MALDI-FISH methods to add to the conversation on brominated metabolite production in marine sponges. Using MALDI-MSI, we measured fistularin-3, a poly-brominated natural product previously isolated from Aplysina aerophoba , present in our A. aerophoba samples that were inoculated with Pseudovibrio brasiliensis . A recent study reported that P. brasiliensis produced fistularin-3 in pure culture, but this data has not yet been reproduced. We employed MALDI-MSI and FISH on the same sponge cryosections to colocalize the spatial distribution of fistularin-3 with P. brasiliensis in sponge tissue. Despite the caveats of this study, our data suggests that perhaps both sponge and microbe may be required for production of fistularin-3.