Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured morphotypes.

, a globally significant N-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of species that form distinct colony morphotypes and are inhabited by diverse microorganisms. colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS), we identified and localized a wide range of small metabolites associated with single-colony morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured morphotypes and provides valuable insights into metabolites common to morphologically distinct colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.