9. Metabolomics

9.1. Introduction

Metabolomics is the study of small molecules, metabolites, and biological intermediate substrates. This omics tool has become very popular within the last decade, including among entomologists (Snart et al., 2015), and applications to understand the biology of honey bees (Ardalani et al., 2021; Broadrup et al., 2019; Chandrasekaran et al., 2015; Chang et al., 2022; Chen et al., 2021; du Rand et al., 2017; Jousse et al., 2020; Klupczynska et al., 2020; Li et al., 2020, 2023; Ma et al., 2024; Paten et al., 2022; Pratavieira et al., 2020; Rand et al., 2015; Ricigliano et al., 2022; Rothman et al., 2019; Shi et al., 2018; Wang et al., 2022; Wu et al., 2024; Wu et al., 2017; Xu et al., 2024; Zhang et al., 2022; Zhao et al., 2020; Zhong et al., 2024), the relationship with their symbionts (Kešnerová et al., 2017; Quinn et al., 2024; Zhang et al., 2022; Zheng et al., 2017), and characteristics of colony products (Arathi et al., 2018; Baky et al., 2023; Chakrabarti et al., 2019; Guo et al., 2020; Koulis et al., 2021; Li et al., 2019; Milone et al., 2021; Qi et al., 2023; Sun et al., 2021; Virgiliou et al., 2020; Wang et al., 2022; Wilson et al., 2013; Yan et al., 2024; Yusoff et al., 2022) are expanding (see Jung (2023) for a brief review). The method allows small molecules to be characterized in a biological system, and is an important complement to more established omics methods, such as genomics, transcriptomics and proteomics. In fact, metabolomics is often considered as the final piece of the omics puzzle (Veenstra, 2012). Metabolomic investigations can be conducted using nuclear magnetic resonance (NMR), capillary electrophoresis mass spectrometry (CE-MS), gas chromatography mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MSMS) (reviewed in Munjal et al. (2022)). LC-MSMS is the most commonly used technique and is the one we focus on here.

Unlike the other omics tools, where species-specific sequence libraries are required, one advantage of metabolomics is that a general library of small molecule fragmentation patterns has been established. Fragmentation patterns and other characteristics (mass, isotope ratio, and retention time) determine the metabolite being identified, rather than a specific sequence of the organism. Metabolomics is thus a widely applicable tool, especially for honey bees, where researchers are trying to assess the biological processes of development or the physiological impacts of various stressors (pesticides, malnutrition, mites, etc.) or other stimuli.

The following general method has been successfully applied to analyze honey bee pollen (Chakrabarti et al., 2019) and royal jelly (Milone et al., 2021), but also works well for conducting metabolite detection in honey bee tissues, where the method has enabled identification of 251 high-confidence metabolites from whole honey bees (Chakrabarti et al., manuscript in preparation). However, as there are many sample types and desired compound classes of potential interest, variations of this protocol may work better for certain applications. For example, while methanol/water extractions are commonly conducted (e.g. Paten et al., 2022; Chang et al., 2023; Xu et al., 2024), extraction solvents composed of a mix of acetonitrile, methanol, and water (e.g. Chen et al., 2021; Zhang et al., 2022; Ma et al., 2024), acetonitrile and methanol (e.g. Liu et al., 2023; Wu et al., 2024), or methanol and chloroform (e.g. Ricigliano et al., (2022)) have also been used for honey bee tissues. The choice of extraction solvent depends on the polarity of the desired metabolites, and additional extraction techniques developed for mammalian tissues (Sitnikov et al., 2016) are likely also applicable to honey bees. For example, in a preprint report, (McAfee et al., 2024) recently applied a two-phase extraction technique (using an initial methanol/water extraction followed by addition of methylated tert butyl ether) developed by (Chen et al., 2013) [1] for mouse liver samples for parallel analysis of the metabolome, lipidome, and pheromone profiles from queen honey bee heads. The following protocol serves as a starting point for researchers interested in conducting metabolomics, with the knowledge that there are many possible variations of the general technique, particularly with respect to the extraction solvent and liquid chromatography solvents.

The method described here is intended for a semi-quantitative (i.e. relative quantitation, which is suitable for differential abundance testing but does not provide information on absolute quantities), untargeted metabolomics approach. These sample preparation guidelines are also applicable to absolute quantitation, provided that the user includes the additional step of creating standard curves for each analyte. However, as untargeted semi-quantitative analysis is the most widely used method, that is the application we focus on for both sample preparation and data analysis methods. Interested readers should familiarize themselves with existing overviews of sample preparation and data acquisition (Broadhurst et al., 2018; David & Rostkowski, 2020; Defossez et al., 2023; Munjal et al., 2022; Rampler et al., 2021), statistical analysis (Bartel et al., 2013; Chen et al., 2022; Xi et al., 2014), and reporting (Alseekh et al., 2021; Sumner et al., 2007) described elsewhere.