Metabolome changes of Megachile rotundata across pupal stages measured by mass spectrometry-based untargeted metabolomics.

Untargeted metabolomics measurements were conducted via Gas Chromatography-Mass Spectrometry (GC-MS) on both sexes, male and female, of the alfalfa leaf cutting bee, Megachile rotundata during three stages: Prepupae (PP), Red Eye Pupae (RE), and Melanized Pupae (ME). Focusing on the analysis of polar metabolites, results uncovered clear metabolic differences between the three stages of M. rotundata. However, metabolic profiles between sexes within each growth stage were indistinguishable. A total of 189 metabolites were tentatively identified between the three pupal stages studied, and these were used to build a relationship between the data and the observations model (growth stage or sex) with Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). Specifically, 50 metabolites showed significant changes between stages PP vs. RE, while between stages RE vs. ME, 70 metabolites showed significant changes (OPLS-DA differentiation where VIP >1 and fold change value > 1.5 between groups), pointing to an increase metabolic activity with growth stage progression. Metabolites associated with the different growth stages studied included amino acids, neurotransmitters, carbohydrates and derivatives (phosphorylated, amino, alcohol), nucleotides and organic acids. Measured relative metabolite levels were rationalized in terms of the prevalent metabolic activities at each developmental stage. The PP stage was characterized by an overall low energy consumption state involving the metabolism of amino acids generated via protein degradation. On the other hand, the ME stage was characterized by high levels of metabolites related to the melanization process (tyrosine, DOPA) as well as phosphorylated carbohydrates, amino acids, nucleic acids and neurotransmitters indicating a state of high energy consumption and structural changes.