Combining Hybridization Chain Reaction–Fluorescence In Situ Hybridization and Multiplexing to Map Salt Marsh Sediments Microbial Community


Paul Rousteau* (, Akshata Shukla, Holly Gustavsen, Jeffrey Marlow


Boston University, Boston, MA



Salt marshes are among the most biogeochemically active environments on Earth. They display an important microbial diversity due to high organic matter input, active tidal cycling, and vegetative colonization. Sequencing technologies have expanded the ability to analyze the microbial population of such complex environments. While it is possible to answer the question of diversity composition, sequencing analysis does not allow researchers to discern the interactions within the microbial community and between the microorganisms and their environment. To this extent, the method of fluorescence in situ hybridization (FISH) is a powerful tool, allowing us, via specific hybridizations, to not only have a visual confirmation of sample diversity, but to observe how the microorganisms are interacting with each other, like symbiosis or predator–prey relationships. Unfortunately, salt marsh sediment poses some technical challenges with the most important being the sediments autofluorescence, making microorganism identification challenging. Furthermore, the intensity of the signal depends on the individual activity of microorganisms. This activity can be low in sediments, and therefore, it can be challenging to observe and differentiate microorganisms within the matrix. Hybridization chain reaction (HCR) FISH is a recently developed technique that allows researchers to observe such diversity with a strong signal while getting over the autofluorescence emitted by the sample matrix by increasing the contrast between labeled microbes and matrix grains. This method is based on the hybridization of initiator probes, targeting a sequence of interest. The presence of initiator probes triggers hybridization with two supplementary labeled probes called amplifiers. The resulting effect increases the signal, causing a higher contrast between the targeted organisms and the matrix. Here, for the first time, researchers used HCR-FISH to identify microorganisms composing the Little Sippewissett microbiome and how they interact within the sediments. The team developed a multilabeling method to efficiently target organisms’ housekeeping genes to identify specific key players in the salt marsh geochemical cycle. The resulting color code allows the team to not only precisely identify microorganisms but shows the interactions between each of them. From those observations, researchers can evaluate the dominance of certain key players in the sediment and how they influence the biogeochemical cycle of salt marsh sediments. This new application of HCR-FISH could be a template for further study based in sediments and could be extended to fill in the gaps between microbial diversity and microbe interactions with its environment.