Effects of pollen diet on Crithidia in bumble bees

Crithidia inoculum

Infected (‘source’) colonies were used to make Crithidia inoculum. The original Crithidia cells infecting colonies came from three wild B. impatiens workers collected from Stone Soup Farm (Hadley, MA, USA: 42.363911 N, −72.567747 W) unless otherwise noted. To make inoculum, bees were dissected from the source colony daily using an established protocol20. Bee digestive tracts (excluding the honey crop) were removed, placed into a 1.5 mL microcentrifuge tube with 300 μL of 25% strength Ringer’s solution (Sigma-Aldrich, St. Louis, MO, USA), finely ground, and vortexed for 5 seconds. Each sample was allowed to rest at room temperature for 4–5 hours. Crithidia cells were counted from a 0.02 μL sample per bee with a Neubauer hemacytometer20. We mixed 150 μL of the supernatant with 25% strength Ringer’s solution to achieve a concentration of 1200 cells μL−1. The inoculum was then mixed with an equal volume of 50% sucrose solution to yield inoculum with 600 cells μL−1 and 25% sucrose. Experimentally infected bees were starved for 4–6 hours and then fed a 10 μL drop of inoculum with 6,000 Crithidia cells, which is within the range of concentrations bees are exposed to when foraging on flowers in the wild52. Only bees that consumed the entire droplet were used in experiments.

Monofloral and mixed pollen

Monofloral pollen diets (rape, sunflower or buckwheat – Brassica campestris, Helianthus annuus and Fagopyrum cymosum, respectively) were obtained by sorting honey bee collected pollen pellets (Changge Hauding Wax Industry, China) initially by color. We then verified microscopically that pollen pellets within treatment were morphologically consistent and as expected for that species. Pollen was provided to bees as a paste made by mixing ground pollen pellets with distilled water to achieve a uniform consistency, which required different amounts of water depending on pollen species (pollen: water ratio: sunflower & buckwheat: 5:1; rape: 1.67:1; pollen mix of equal weights of the three monofloral pollens: 3.33:1).

Newly emerged adult worker bees (callows) obtained from pupal clumps were removed from six uninfected B. impatiens colonies (n = 272 bees). All B. impatiens colonies were provided by BioBest LTD (Leamington, Ontario, Canada), and experimental colonies were confirmed to be pathogen-free bi-weekly by screening five workers (see Crithidia inoculum). We regularly supplied all colonies with pollen loaves made of 30% sucrose solution mixed with ground honeybee–collected wildflower pollen (Koppert Biological Systems; Howell, MI, USA). Each day, newly emerged callows were collected from pupal containers, weighed to the nearest 0.01 mg, and randomly assigned to one of the four pollen diets. Bees were randomly assigned to treatment within experimental colony and, when relevant, date of emergence, for all experiments here and below. Bees were housed individually in a growth chamber in darkness at 28 °C and fed 500 μL of 30% sucrose solution and a small ball of their respective pollen treatment daily for 9 days. Bees were inoculated two days after emergence, so that bees consumed their respective pollen treatments both before and after infection.

Crithidia infection intensity was measured as Crithidia cells per 0.02 μL (hereafter “cell counts”) one week after bees were infected (n = 234 bees due to mortality). After 7 d, Crithidia infection intensity reaches a sufficient level for measurement within the bee host53. Each experimental bee was dissected (see Crithidia inoculum). We removed the right forewing of each bee and mounted them on glass slides to measure radial cell length, a proxy for bee size54.

Consistency with a different pathogen strain

Crithidia infection can be heavily influenced by genotypic variation in hosts and pathogens55, which may yield genetically distinct strains with varying susceptibility to host immune defenses56 and potentially responses to pollen diet. Thus, we repeated our experiment testing the effects of pollen diet on a different set of colonies infected with a strain obtained from wild B. impatiens collected in Raleigh, North Carolina, USA (J.C. Roulston Arboretum: 35.794056 N, −78.698186 W). Given the strong negative effects of sunflower pollen on Crithidia (see Fig. 1A), we used only sunflower pollen (H. annuus) and buckwheat pollen as our control (F. cymosum). In addition, adult workers (rather than newly emerged callow bees) were used in this experiment to ensure that results were consistent across bees of varying ages. Worker bees were used from three colonies, and bees were inoculated and Crithidia pathogen loads were measured (n = 149 bees).

Effect of diet post-infection

We tested whether sunflower pollen could reduce Crithidia infection in bees that already reached sufficient infection levels. Individual B. impatiens adult workers from three colonies were inoculated with Crithidia (North Carolina, USA strain) and fed a wildflower pollen mixture (Koppert Biological Systems; Howell, MI, USA) and 30% sucrose solution for 7 days. Each bee was then randomly assigned to one of three pollen diets: sunflower, buckwheat, or the same wildflower mix for 7 more days. By including a wildflower mix pollen treatment, we were able to compare monofloral pollen treatments to a more natural and diverse mix of pollens. Bees were then sacrificed (n = 74) and Crithidia pathogen loads were measured.

Consistency using two sources of sunflower pollen

Domesticated sunflower is a major oil crop distributed worldwide35. Breeding practices have modified a wide array of economically important traits, including seed and oil production57, resistance to plant diseases and pests58, and resistance to drought59. We compared the medicinal effects of sunflower pollen from China versus sunflower pollen from the USA. Adult B. impatiens workers from three colonies were inoculated with Crithidia (n = 120 bees) and fed either sunflower pollen collected from an organic farm in Wisconsin, USA (44.731641 N, −91.948666 W, Cobalt II cultivar - NuSeed Inc.), sunflower pollen collected in China (Changge Hauding Wax Industry, China), or the wildflower pollen mixture. We measured pathogen loads (n = 110 bees) after 7 days.

Statistical analyses

All statistical analyses here and below were conducted using R version 3.1.260 (Supplementary Information: Methods 1). To test how pollen diets affected Crithidia infection intensity, generalized linear mixed models were used to analyze Crithidia cell counts using “glmmTMB”61, with pollen diet as a fixed effect, bee size as a covariate, and experimental bee colony and inoculation date (if applicable) as random effects. Significance of terms was evaluated with a likelihood ratio chi-squared test, implemented via the “drop1()” function. Tukey’s HSD tests were used for post hoc pairwise comparisons. All bees that died before their scheduled dissection date were excluded from analyses. To test how pollen diets affected bee survival, mixed-model Cox proportional hazards tests were used62, with pollen diet and bee size as fixed effects, and inoculation date (if applicable) and experimental bee colony as random effects. To assess the effects of pollen diet on mortality, log-likelihood of models were compared with and without pollen diet treatment as a predictor. Significance of terms was tested with a Wald chi-squared test, implemented via the Anova function in package “car”63. Plots (here and throughout) were produced with ggplot264, survminer65 and cowplot66.

Costs and benefits of sunflower pollen for bee health, reproduction and Crithidia

Using queenless B. impatiens microcolonies, we tested the impact of pollen diet and Crithidia infection on mortality, reproduction and Crithidia infection in a 2 × 2 factorial design manipulating pollen diet (sunflower or buckwheat) and Crithidia infection (uninfected or infected). When unmated workers are isolated from the queen, one will gain dominance and lay haploid (male) eggs. Microcolonies are an effective approach to estimate the effects of diet and pathogen infection on whole-colony reproduction15,20,67. We used 20 replicate microcolonies per treatment for a total of 80 microcolonies, carried out in two rounds (or blocks) of 40 microcolonies, with five workers per microcolony. The first 40 microcolonies were constructed using workers from two colonies, with 5 replicates per treatment per colony of origin. The second set of 40 microcolonies were constructed from two new colonies of origin.

Microcolonies were randomly assigned to infection and diet treatments within rounds and colonies of origin. Bees were inoculated with Crithidia as in ‘Crithidia inoculum’ or given a sham control inoculum of 10 µL of sucrose solution without Crithidia cells. We maintained microcolonies in a growth chamber at 28 °C in darkness and fed them 400 mg of pollen each and ad libitum 30% sucrose solution, replaced and replenished 5 d per week. Pollen diets were made as in ‘Monofloral and mixed pollen’. We measured pollen and sucrose solution consumption (in g) 5 days per week, calculating the total mass consumed (or used) per bee per hour. Pollen consumption was corrected for evaporation by subtracting the average weight lost to evaporation over 24 hr for each pollen type. To determine the average weight lost to evaporation, 15 samples of each pollen type were placed into empty microcolony containers without bees and in the same growth chamber for 24 hr. Each pollen sample was weighed at 0 hr and at 24 hr to determine the net weight lost to evaporation.

For each microcolony, we recorded the date of first eggs laid, male emergence and weight (which occurred in 5 of the 80 microcolonies) and worker mortality. Microcolonies were terminated 35 days post-egg laying, or if 4 out of the 5 worker bees died. We then measured Crithidia infection in the remaining worker bees (see Crithidia inoculum) and bee size. For each microcolony, the number of eggs, larvae, and pupae produced was counted and weighed. Because bees within microcolonies can vary in size and social dominance, which can affect food consumption and microcolony reproduction, we calculated a metric of within-microcolony size dimorphism [(largest bee radial cell/smallest bee radial cell) − 1]20,68,69 for use as a covariate.

To analyze pollen and nectar consumption, Crithidia infection intensity, and microcolony reproduction, generalized linear mixed effects models were fit with distributions specific to the type of data analyzed (Supplementary Information: Methods 2). Unless otherwise noted, all models included fixed effects of pollen diet (sunflower or buckwheat), infection treatment (infected or uninfected), and (when significant) their interaction. All statistical tests included block as a random effect, which corresponded to microcolonies inoculated on the same day. The block effect accounted for variation due to colony of origin (because each inoculation day used a different colony of origin) and variation due to different inoculation dates.

Effects of pollen diet on Nosema in honey bees

Newly emerged worker honey bees from three colonies were mixed together and placed into cages in groups of 50 bees per cage70 with 50% sucrose solution. We experimentally infected the bees in each cage using a Nosema spore sucrose solution with a concentration of approx. 333,333 spores per bee71,72. Cages were randomly assigned to a pollen diet treatment and given a single 20 g ball of sunflower or buckwheat pollen paste, or no pollen as a negative control for 15 days. Prior studies have shown that Nosema-infected honey bees that do not consume pollen have significantly lower Nosema infection intensity than bees provided with pollen71. There were 11–12 replicate cages per pollen diet treatment. On days 10 and 15, samples of five bees and 10 bees per cage, respectively, were sacrificed to quantify Nosema infection intensity71,72. Any bees that died during the experiment were counted and removed from their cages.

We used generalized linear mixed effects models (R package glmmTMB) to test whether pollen diet affected Nosema infection intensity (spores per mL) on days 10 and 15 (Supplementary Information: Methods 3). Nosema infection intensity was used as the response variable; pollen treatment, days since inoculation, and their interaction were used as fixed predictors; cage was included as a random effect to account for repeated measures on each cage. Differences in survival were tested using a Cox Proportional Hazards mixed-effects model fit using “coxme”62, with pollen diet as a fixed effect and cage as a random effect.

Effect of sunflower plantings on Crithidia infection in bumble bees at the farm scale

Bees were collected directly from sunflowers if available, or else from a variety of flowering crops. Each farm was sampled on a single date. We quantified the area of sunflower grown at each farm in m2. We sampled a total of 667 B. impatiens workers (range: 19–62 bees per farm); all bees were sacrificed and we measured Crithidia infection (as in Crithidia inoculum).

We tested for spatial autocorrelation using a Monte-Carlo Mantel test and a Moran’s I test using the “ape” and “ade4” packages in R73,74. We found no indication of spatial autocorrelation (P > 0.15), and so considered farms to be independent sampling locations. We analyzed infection intensity (Crithidia cell counts) with a generalized linear mixed model with negative binomial error distribution using the “glmmTMB” package in R61. Sunflower area (log 10 area (m2)) and Julian date of sampling were used as fixed covariates; farm was included as a random effect to account for non-independence of bees within a farm. Sampling date and sunflower area were not confounded (Pearson’s r = 0.02). Significance of predictors was tested by likelihood ratio chi-squared tests, implemented via the “drop1” function in R.

Pesticide analysis

To ensure that results were not associated with pesticide residues on pollen, the USA and Chinese sunflower, buckwheat, and the wildflower mix pollens were analyzed for 213 pesticides and other agrochemicals (Agricultural Marketing Services’ National Science Laboratories, United States Department of Agriculture, Gastonia, NC USA) (Supplementary Information: Table 1).