Metabolomics Enables the Structure Elucidation of a Diatom Sex Pheromone

Abstract

Diatoms are unicellular photosynthetic organisms that often dominate primary production in pelagic and benthic aquatic ecosystems. Despite their central role in the biosphere, little is known about their pheromone chemistry and their lifecycle, which is characterized by asexual population growth alternating with short bursts of sexual reproduction. Diatoms are unique among microalgae in that sexual reproduction is only possible below a species-specific sexual size threshold (SST). This SST is intimately linked to the characteristic cell division of diatoms. Due to their rigid biomineralized cell wall, mitotic division results in a reduction in size (Figure 1). Size is restored typically by sexual reproduction. Upon germination, a zygote generates a large initial cell, which begins a new round of vegetative proliferation. In the ancestral group of predominantly planktonic centric diatoms, environmental cues induce meiosis in cells below the SST, resulting in the formation of eggs and flagellated sperm. However, in the youngest and most species-rich pennate raphid diatoms, which have adopted a primarily benthic lifestyle, it is the pairing of diploid cells that triggers the production of isogametes. Although the SST is known to control the mating capacity of pennate diatoms and indirect evidence suggests the involvement of pheromones, the regulatory principles underlying the differentiation of mating cells and the identity of the pheromones remain unknown. We studied the physiological and metabolic changes associated with sexual reproduction in Seminavis robusta, a model for studies of pennate diatom lifecycles, with the goal to elucidate its pheromone chemistry. Mating is strictly size-dependent with a narrowly defined SST of (51.6 0.5) mm (Figure 2a). Like most pennate diatoms, S. robusta is a heterothallic species, that is, a species with morphologically identical but physiologically distinct cell types between which fertilization can take place. Mixing G1 phase-synchronized cells below the SST revealed the physiological differences between cells of different mating types (Figure 2b). Depending on the density of the partners, pairs or clusters of a migrating mating type (designated MT) around an attracting cell (designated MT ) were observed. The increased motility of MT in the presence of medium from a mating culture suggested the involvement of regulatory pheromones. We developed a bioassay to unambiguously prove and quantify the effect of such pheromones. Therefore hydrophilic/lipophilic-balanced solid-phase extraction cartridges (HLB-SPE) were loaded with media of S. robusta cultures. The SPE absorbent beads were then removed from the cartridges and served as pheromone sources. Behavioral responses towards such beads were monitored using light microscopy. Beads loaded with medium from amating culture attracted MT cells below the SST proving that the pheromone can be extracted (Figure 2b, movie in the Supporting Information). Attraction by MT cells was dependent on cell size and on prior perception of sexually mature (i.e. below the SST) mating partners. Extracts of MT cultures below the SST were only active when the MT cells were previously conditioned with medium from MT below the SST (Figure 2c). Extracts of MT above the SSTwere not active, even after conditioning with medium from MT below the SST (Figure S1 in the Supporting Information). Likewise, theMT motility response is primed by MT signals. Attraction to pheromone-loaded beads only occurred if MT cells were