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

Authors: MONDAL, BANANI - University Of Wyoming; WALTER, RIKKI - North Dakota State University; Rinehart, Joseph; GREENLEE, KENDRA - North Dakota State University; BASILE, FRANCO - University Of Wyoming

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2026
Publication Date: 2/10/2026
Citation: Mondal, B., Walter, R., Rinehart, J.P., Greenlee, K., Basile, F. 2026. Metabolome changes of Megachile rotundata across pupal stages measured by mass spectrometry-based untargeted metabolomics. Insect Biochemistry and Molecular Biology. https://doi.org/10.1016/j.ibmb.2026.104513.
DOI: https://doi.org/10.1016/j.ibmb.2026.104513

Interpretive Summary: The alfalfa leafcutting bee is an important pollinator of agricultural crops including alfalfa for seed production and canola, among other crops. As the most intensively managed solitary bee, it is crucial to understand this insect’s underlying biological processes to ensure that management practices are optimized for bee survival and improved health. While several studies have focused on metabolic rates, gene expression profiles, and other parameters, there has been a lack of studies on how these parameters interact. This interaction can best be seen by changes in the metabolome: the group of small molecules created and destroyed during metabolic processes. In this project, we assessed the metabolome of alfalfa bees in three different stages of pupal development. We found a total of 289 metabolites, with differences discernable amongst the different developmental stages. This will serve as a baseline for future studies on the biology of this important alternative pollinator.

Technical Abstract: 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 pupae stages of M. rotundata. However, metabolic profiles between sexes within each growth stage were indistinguishable. A total of 289 metabolite entities or features 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, 38 metabolites showed significant changes between the stages PP vs. RE, while between stages RE vs. ME, 65 metabolites showed significant changes (OPLS-DA differentiation where VIP >1 and fold change value > 1.5 between groups and P-value < 0.05). Metabolites associated with the different growth stages studied included amino acids, neurotransmitters, sugar phosphates, amino sugars and sugar alcohols. Interestingly, not all the amino acids detected followed the same trend with growth stages. For example, isoleucine, tyrosine, serine, threonine, valine, asparagine, and alanine were upregulated in the ME group (when compared to both RE and PP) with an overall trend of increasing relative concentrations from PP to the ME stage. On the other hand, hydroxyproline was upregulated in the PP group and decreased in relative concentration with growth stage (i.e., PP > RE > ME). Increased levels of these amino acids in pupae may be attributed to their role as the main building blocks of proteins during this period of growth & development. Conversely, the GC-MS analysis of the lipid acyl groups (as their fatty acid methyl esters) showed no significant changes with the sex of the pupae or between the growth stages.