Location: Honey Bee Breeding, Genetics, and Physiology Research
Project Number: 6050-21000-016-025-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Dec 1, 2022
End Date: Dec 31, 2023
Objective:
Our objective is to isolate and identify phagostimulants from pollen that could be used to improve the attractiveness and palatability of artificial “pollen substitute” diets for honey bees. Using tools and skills that are well established in the natural products research community, we will first extract different pollens using a variety of solvents, characterize the chemical composition of these fractions using mass spectrometry, and then test their effectiveness using honey bee choice assays in the laboratory and in field colonies. Next, we will isolate and chemically characterize the most attractive fractions to identify specific compounds that are responsibly for pollen’s phagostimulatory effects. Purified compounds will be isolated on a larger scale or, where possible, purchased from chemical supply companies to recapitulate the most efficacious samples.
Approach:
1. A series of solvent extractions, within an increasing polarity gradient be carried out on samples of bee-collected, trapped pollens. Extractions will be performed in series on the same pollen samples.
2. The three resulting extracts will be analyzed by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The GC-MS conditions will follow literature precedent. The LC-MS conditions will use well established techniques of our collaboration [16]. These data will be analyzed based on accurate mass and MS/MS fragmentation patterns, using both literature precendent and mass spectrometry prediction software . In our recent study, we detected a broad spectrum of pollen metabolites. We expect the number of detectable metabolites to markedly increase with an optimized extraction protocol that is being developed using natural products chemistry expertise.
3. Following metabolomic analyses, the fractions will be dried and added to a base artificial diet as a control or a substrate to which pollen extract fractions can be added for phagostimulant activity treatments.
4. For honey bee choice assays in laboratory cages. The diets infused with different pollen extract fractions will be presented in order to establish the most attractive formulations. First, all diets containing pollen fraction extracts will be tested against the control diet (no pollen extract). Then, all permutations of binary choice assays among the diets will be conducted. For example, diet A versus diet B will be presented to the same cage of bees, and consumption will be weighed and replenished over the course of 9 days (Figure 1). The preferred diet will then be assayed against others via cage tests until a preference hierarchy has been established. Diet samples in empty cages will be used to calibrate moisture loss due to evaporation in the diets, and consumption measures will be adjusted accordingly.
5. Diets containing the most attractive pollen extract fractions will be scaled up to in-hive choice assays. Patties will be prepared as noted above and presented in the same hive using three hive replicates per test. Similar to laboratory assays, we will test all permutations of the diets in binary choice assays (Figure 1).
6. The most promising diets, and therefore the most attractive pollen fractions, will be subjected to spectrometric and spectroscopic analyses to identify the most prominent compounds present in the fractions. The isolation of these compounds will be scaled up, as needed, using well established protocols [19-21], or where possible, these compounds will be purchased from chemical supply houses.
