Pseudo-nitzschia multistriata is a planktonic marine diatom with a diplontic life cycle comprising a short sexual phase, during which gametes are produced following the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Herein, we have hypothesized that the amount and nature of cellular metabolites varies along the mating process. To capture the metabolome of Pseudo-nitzschia multistriata at different harvesting times in an unbiased manner, we undertook an untargeted metabolomics approach based on liquid chromatography–tandem mass spectrometry. Using three different extraction steps, the method revealed pronounced differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT−) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; additional 46 features could be classified by molecular networking of tandem mass spectra. The reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. Moreover, the role of highly regulated compounds such as 7-dehydrodesmosterol, whose changes in abundance were the highest in the experiment, oleamide, ectoine, or trigonelline is discussed.

Diatoms are photosynthetic microorganisms playing a key role in the functioning of aquatic ecosystems; they are at the base of the food web and are the main drivers of biogeochemical processes. These microalgae have a unique diplontic life cycle in which the vegetative phase entails a cell size reduction that would lead to the extinction of the cell population if the size was not restored, usually by sexual reproduction. The switch from asexual to sexual reproduction needs to be finely synchronized and regulated to ensure its success; to this aim, cells evolved complex chemical crosstalk that mediates mating. We focused our attention on the marine diatom Pseudo-nitzschia multistriata, investigating the reciprocal perception of the opposite mating type (MT) and the genes and signaling molecules putatively involved in the process. From previously available transcriptomic data, we selected a panel of genes deregulated during the early phase of sexual reproduction, confirming for some of them a role during mate perception and establishing a hierarchy governing their behavior. Moreover, we explored the nature of the molecules controlling sexual reproduction in this species, unveiling that the signaling is mediated by the secretion of protein and non-protein cues from the MT− and MT+, respectively. Our results point to a model in which the two MTs stimulate each other, but the stimulation by the MT− is amplified after MT+ perception in a positive feedback manner.