Background

Bumble bees are valuable pollinators in agricultural and natural settings (Kleijn et al., 2015). Disconcertingly, populations of some bumble bee species are in serious decline (Cameron et al., 2011). Many factors are believed to contribute to the reported population declines, including poor nutrition, parasites, pathogens, and pesticides (Brown and Paxton, 2009; Goulson, 2005, 2013, 2015; Meeus et al., 2011; Wood et al., 2019). Recognizing their importance and the number and complexity of factors affecting their populations, there is a need to better understand bumble bee biology and the effects of environmental stressors on bumble bees.

Microcolonies are formed when a small group of bumble bee workers is isolated in a queenless environment. Under these conditions, the workers self-organize to build nest structures and lay unfertilized eggs that produce drones (Free, 1955). The model is versatile, offering the ability to investigate a range of endpoints, including behavior, the gut microbiome, nutrition, development, pathogens, and pesticide exposure (reviewed in Klinger et al., 2019).

Currently, there are no detailed protocols for initiating and monitoring microcolonies published for any bumble bee species, only condensed protocols in the methods sections of research publications (Gradish et al., 2012, 2013; Smagghe et al., 2007). Here, we detail a step-by-step protocol for initiating and monitoring bumble bee microcolonies with the common eastern bumble bee (Bombus impatiens Creson) (Hymenoptera: Apidae). We also provide detailed instructions for preparing microcolony food provisions. An overview of the procedures for initiating and monitoring microcolonies can be found in Figure 1. The protocols presented here were originally described in two peer-reviewed publications (Camp et al., 2020a, 2020c) and a subsequent publication comparing these two studies (Weitekamp et al., 2022). While these protocols were designed for assessing the effects of pesticide exposure on bumble bees, they can be easily expanded to investigate other aspects of bumble bee biology, including behavior, nutrition, development, pathogens, and gut microbiome (reviewed in Klinger et al., 2019).

Figure 1. Overview of procedures for initiating and monitoring microcolonies. (A and B) Prepare syrup and pollen stocks for provisioning the microcolonies. Although syrup can be prepared in advance and stored at 4°C, pollen patties should be made fresh on the day of use. Transfer pollen to dishes and collect the weight. (C) Use only age-matched, newly emerged B. impatiens workers when using this protocol. To facilitate experimental manipulation, chill workers on ice. Distribute five bees to each microcolony chamber. Provision microcolony chambers with a ~3 g pollen patty for nest building and a syringe feeder filled with 50/50 inverted syrup. Supplement the nest with ~2 g of pollen paste on day 5. (D) Provide microcolonies with pollen patties for feeding (starting on Day 7) and 50/50 inverted syrup every Monday, Wednesday, and Friday for the duration of the experiment (recommend no more than 49 days). Collect the weight of the old syringe feeders and pollen dishes to use when calculating food consumption. (E) Investigators are encouraged to collect data on worker mortality and drone production (i.e., timing to emergence of 1^st drone, number of drones emerged, and drone weight). Syrup and pollen consumption values should be corrected for evaporation and worker mortality. The black vertical arrow on the righthand side indicates the order of operations for initiating and monitoring microcolonies.

Part I: Protocol for microcolony food preparation