Fig 1.
Annual cycle of social bumblebees.
The cycle begins when mated queens emerge from their overwintering sites in early spring and search for suitable nest sites. Once a suitable nest site has been found, the queen collects and stores floral resources (nectar and pollen) to raise the first brood of workers, which emerges in late spring. A subset of the workers emerging become ‘foragers’ and leave the nest to collect floral resources for the queen and the rest of the workers in the colony. The number of workers (foragers and nest bees) in the colony continues to grow over the summer months. From late summer into the fall, the colony starts to produce new queens and males (reproductives) instead of workers. Reproductives leave the nest soon after emergence and search for a suitable mate until late fall. Once mated, queens search for an overwintering site and once located, reside there until the spring. Males and colonies (founding queen and workers) do not survive the winter, thus completing the cycle.
Fig 2.
Schematic of setup for individual feeding assay.
Bees were isolated from the colony and housed in a 16oz plastic container with a feeding cup positioned directly under an open vent in the screen lid. Test sugar solutions were delivered through a hole in the lid with a micropipette. Individuals were supplied with pollen ad libitum.
Fig 3.
Kaplan-Meier survival plots for Bombus impatiens (A) queens, (B) workers, and (C) males, orally exposed to test solutions containing different concentrations of clothianidin daily for 7 days. Black = 0ppb; red = 5 ppb; green = 7 ppb; blue = 10ppb. A Mantel-Cox Log-rank analysis was used to test for differences in survival between each treatment group relative to the 0ppb control group over the 7-day period. **, p<0.0001.
Table 1.
Response of queen, worker, and male bumblebees (Bombus impatiens) to chronic clothianidin exposure controlling for differences in body size and daily dose.
Workers and males consumed 75 μL of solution per day and due to their larger size, queens consumed 200 μL of each solution per day. Yellow shading = no effect on survival; blue shading = reduced survival. All values are shown as mean ± SD.
Fig 4.
Heatmap showing the 25 putative detoxification genes that were differentially expressed between workers and males after 5 consecutive days of oral exposure to clothianidin.
Phase 1 (P1) detoxification processes include oxidation, hydrolysis, and reduction reactions while Phase 2 (P2) processes include conjugation reactions. Legend on the right shows relative fold change corresponding to each color.
Table 2.
Sex-specific expression of inducible detoxification genes in bumblebee workers and males orally exposed to clothianidin at a sub-lethal daily dose over 5 consecutive days.
Phase 1 detoxification processes include oxidation, hydrolysis, and reduction reactions while Phase 2 processes include conjugation reactions. Expression levels were determined through RNAseq analysis. Upper portion = genes with increased expression in workers; lower portion = genes with increased expression in males. Clothianidin-inducible genes were initially identified by comparing transcriptomic expression patterns between individuals fed either 5ppb clothianidin solution or the same volume of solution containing 1.4% DMSO (vehicle control) solution separately for each sex and then pooling genes with significantly altered expression together.