The effect of sunflower pollen age and origin on pathogen infection in the common eastern bumble bee (Apidae: Hymenoptera)

Abstract

Bumble bees are globally important pollinators, contributing hundreds of millions of dollars annually in crop pollination services. Several species are in decline, making it paramount to understand how pathogens and nutrition shape bee health. Previous work has shown that consuming sunflower pollen (Helianthus annuus) dramatically reduces infection by the trypanosomatid gut pathogen, Crithidia bombi, in the common eastern bumble bee (Bombus impatiens). Sunflower pollen may therefore be useful as a dietary supplement for reducing this pathogen in managed bumble bee colonies. Here, we assessed the efficacy of freezer-stored sunflower pollen that was collected in different years and locations for reducing pathogen infection. We tested sunflower pollen that was 1, 3, 4, or 5 yr old and from sunflowers grown in the United States or China against a control of 1-yr-old buckwheat pollen from China, since buckwheat pollen results in high infection. We hypothesized that older pollen would have weaker medicinal effects due to degradation of pollen quality. We found that all sunflower pollen treatments significantly decreased Crithidia infection compared to controls. These results suggest that sunflower pollen can be freezer-stored for up to 5 yr and sourced from a wide range of geographic areas and still maintain its medicinal effects against Crithidia in the common eastern bumble bee. This is helpful information for stakeholders who might administer sunflower pollen as a dietary supplement to manage Crithidia in commercial bumble bee colonies.

Introduction

Bees are essential pollinators for wild and agricultural ecosystems, yet many species are in decline due to combined stressors such as resource scarcity and pathogens (Goulson et al. 2015). Bumble bees are some of the few bee species that have been commercialized in recent decades and commercial colonies now provide millions of dollars toward agricultural pollination annually (Velthuis and Doorn 2006). Colonies are typically reared in large facilities and shipped to buyers for pollinating in farms or greenhouses. These bees are susceptible to additional stressors such as frequent transportation, temperature fluctuations, and high densities, leading to high pathogen risk (Cameron et al. 2016). Pathogens can spread rapidly through a commercial facility and cause further issues by spreading to wild bees in nearby landscapes (Colla et al. 2006). Therefore, reducing pathogens in bee-rearing facilities and commercial colonies is paramount for the welfare of both commercial bees and surrounding wild bee populations.

Recent research found that pollen from sunflowers (Helianthus annuus) dramatically reduced infection levels of a gut pathogen, Crithidia bombi, in the common eastern bumble bee, Bombus impatiens (Giacomini et al. 2018), which is the dominant commercial species in North America. Bumble bee-rearing companies and buyers may consider using sunflower pollen as a dietary supplement to reduce Crithidia infection in colonies. However, further information is needed to implement the use of sunflower pollen supplements, such as its shelf life and the breadth of this effect across sunflower cultivars.

Here, we tested the effect of sunflower pollen collected 1, 3, 4, and 5 yr ago from farms in the United States and China, on Crithidia infections in B. impatiens. Based on previous work that found many cultivars of sunflower were similarly effective against Crithidia (LoCascio et al. 2019), we hypothesized that pollen produced by plants from different locations would be equally effective. We also hypothesized that chemical characteristics of pollen would degrade over time, resulting in decreased medicinal effects in older pollen. This research aims to improve our understanding of how sunflower pollen can be used to manage bumble bee pollinator health.

Methods

Study System

The common eastern bumble bee, B. impatiens (Apidae), is abundant in the wild (Cameron et al. 2011) and commercially available in the eastern United States (Velthuis and Doorn 2006). We purchased 3 B. impatiens colonies from Koppert Biological Systems (Howell, Michigan, USA). Colonies were fed a mix of wildflower pollen (CC pollen, Glendale, AZ, USA) and 30% sucrose solution, and kept in darkness at 27 °C. Crithidia bombi (Trypanosomatidae) is a gut pathogen commonly found in bumble bees that spreads by ingesting contaminated feces (Durrer and Schmid-Hempel 1994). Crithidia can negatively affect bumble bee colony fitness by reducing worker survival and colony founding success by queens (Brown et al. 2003, Fauser et al. 2017). The Crithidia cells in this experiment were originally obtained from 3 wild B. impatiens workers from Stone Soup Farm in 2014 (Hadley, MA, USA) and maintained in the lab in commercial B. impatiens colonies.

Sunflower (Helianthus annuus, Asteraeceae) is a native North American wildflower and major oilseed crop worldwide. Its pollen dramatically reduced Crithidia infection in B. impatiens (Giacomini et al. 2018). This effect is consistent across multiple Crithidia strains (Giacomini et al. 2018, Fowler et al. 2022a) and sunflower cultivars (LoCascio et al. 2019). We purchased sunflower pollen from several locations in both China and the United States across multiple years (Table 1). We used buckwheat (Fagopyrum esculentum, Polygonaceae) as a control because, compared to sunflower pollen, it has similar protein levels, but results in higher Crithidia infection in B. impatiens (Giacomini et al. 2018). We purchased buckwheat pollen from one of the same Chinese companies and in the same year as the 1-yr-old sunflower pollen (Table 1). All pollens were originally collected by honey bees and therefore were a mix of pollen and nectar. Pollens were ground and mixed with distilled water to make a paste that remained frozen at −20 °C until use.

Experimental Design

We selected worker bees from each of the 3 colonies, placed them in individual vials, and inoculated them with Crithidia. Bees were randomly assigned to pollen diet treatments, which they received for 7 days, and were then dissected to assess pathogen infection. We prepared Crithidia inoculum on each trial date by diluting 150 μl of homogenized gut solution from a single infected bee with Ringer’s solution and then adding an equal amount of 50% sucrose, for a final concentration of 600 cells/μl and 25% sucrose. Each bee received a 15-μl inoculum drop (9,000 Crithidia cells) and was observed until the drop was fully consumed. Once inoculated, we transferred bees into individual 16-oz plastic deli cups with mesh bottoms and lids with holes. Bees were provided ad libitum access to 30% sucrose solution and ~1.5 g of their pollen treatment, replaced every other day. Bees were kept in darkness at 27 °C.

We measured pollen and sucrose consumption of each bee over a 48-h period. We weighed the pollen provision and sucrose solutions before and after giving it to each bee. We measured evaporation of each pollen diet by including deli cups with pollen and sucrose solutions but no bees (n = 10 per treatment). To account for evaporation, we built a linear regression of final pollen weight predicted by initial pollen weight for the controls of each pollen treatment (no bees), with the intercept set to 0 to estimate predicted final pollen weights after evaporation. We then subtracted the observed final pollen weight from the predicted final pollen weight for each experimental replicate to estimate consumption.

After 7 days, we dissected and placed individual guts into 1.5-ml microcentrifuge tubes with 300 μl of Ringer’s solution. We homogenized each gut using a tissue grinder and left it at room temperature to settle for 4 h. We then placed a 10-μl aliquot of the supernatant on a hemocytometer and used a compound light microscope at 400× to count moving cells in 0.02 μl of the gut solution. We removed the right forewing from each bee and measured marginal cell length to estimate bee size (Nooten and Rehan 2020). We entered 240 bees into the experiment across 10 start dates between 31 May and 21 June 2021. One bee escaped and one bee died, resulting in 238 bees.

Statistical Analyses

We used R version 4.2.1 (R Core Team 2022). We selected and report results from top models based on AIC, always retaining the pollen diet treatment since it is our variable of interest. To analyze Crithidia cell counts, we used a generalized linear mixed model (glmmTMB function) with a negative binomial distribution (Brooks et al. 2017). We included pollen treatment (8 levels, encompassing age, origin, and type into one factor) and marginal wing cell length as fixed effects and inoculation date as a random effect. We then conducted a Tukey’s Honestly Significant Difference test with the multcomp package to assess all pairwise comparisons between pollen diets (Hothorn et al. 2008).

To analyze pollen and sucrose consumption, we used separate linear models with a normal distribution and pollen treatment and marginal wing cell length as fixed effects. We first log-transformed sucrose consumption to meet model assumptions. Mortality in the experiment was very low (<1%), and therefore we did not analyze survival.

Results

Pollen treatment significantly affected Crithidia cell counts (χ² = 84.55, df = 7, P < 0.0001). Bees fed sunflower pollen, regardless of age or origin, had significantly lower Crithidia counts than bees fed buckwheat pollen (|z| > 5.08, P < 0.001 for all sunflower-buckwheat pairwise comparisons; Fig. 1). On average, sunflower-fed bees had 94.8% lower counts than those fed buckwheat. Bees fed 3-yr-old Chinese sunflower pollen had particularly low Crithidia counts, which were significantly lower than all other pollens (|z| > 3.05, P < 0.04 for all pairwise comparisons) except for the 3-yr-old US sunflower pollen (pairwise comparison: z = 2.73, P = 0.11). Bee size had a marginal effect on Crithidia counts, with smaller bees having slightly higher cell counts (χ² = 3.73, df = 1, P = 0.05). Pollen consumption was not significantly affected by treatment (F = 1.57, df = 7, P = 0.15). Sucrose consumption was marginally affected by treatment (F = 1.98, P = 0.06); however, there were no significant differences across pairwise comparisons after adjusting for multiple comparisons using Tukey’s Honestly Significant Difference test. Larger bees consumed more pollen and sucrose than smaller bees (F > 18.83, P < 0.0001 for both).

Discussion

Consistent with previous work, sunflower pollen dramatically reduced Crithidia infection in B. impatiens workers (Giacomini et al. 2018, 2021, Fowler et al. 2020). Furthermore, sunflower pollen from both the United States and China were effective against Crithidia infections, consistent with our prediction and a previous study that found 15 sunflower pollen cultivars from China, Germany, and the United States all suppressed Crithidia infections compared to buckwheat pollen (LoCascio et al. 2019). It appears this effect against Crithidia is widespread in the Asteraceae family; another recent study found that pollen from multiple other Asteraceae species including goldenrod (Solidago sp.), dandelion (Taraxacum officinale), ragweed (Ambrosia artemisiifolia), cocklebur (Xanthium strumarium), and dog fennel (Eupatorium capillifolium) also reduced Crithidia infections when ingested by B. impatiens (LoCascio et al. 2019, Figueroa et al. 2023).

We additionally found that sunflower pollen can maintain its efficacy against Crithidia after being stored for up to 5 yr at −20 °C. We predicted that pollen quality would degrade over time and that older pollen would be less effective against Crithidia, based on our initial hypothesis that the medicinal effect of sunflower pollen is due to chemical components. However, to date, all of the sunflower chemical components we have assessed (secondary compounds, fatty acids, and extracted metabolites) were not effective against Crithidia when mixed with control pollen (Adler et al. 2020, Figueroa et al. 2023). Rather, after completing the current study, we discovered that the spiny pollen exine appears to be responsible for the effect (Figueroa et al. 2023), possibly by disrupting Crithidia cell attachment to the gut wall. Given that sunflower pollen disrupts Crithidia infection by a physical rather than chemical mechanism, it makes sense that the medicinal effect was maintained over time since the exine structure does not degrade. After all, pollen structure is so resistant to degradation that pollen grains recovered from soil cores can be used to identify plant communities from thousands of years ago (Rozema et al. 2006).

We did not assess chemical or nutritional degradation of the pollen in this study, so it is possible that pollen chemistry was altered by microbial degradation. However, sunflower pollen has notable antifungal properties due to its hydroxycinnamic acid amides (Kyselka et al. 2018), suggesting that it may be more resistant to degradation than other pollen types. Given the high survival rates, we found no obvious negative effects of feeding the bees older pollen of either species.

Sunflower pollen (and the other Asteraceae species listed) may be beneficial as a dietary supplement to reduce Crithidia infections. However, Asteraceae pollen has relatively low protein and sodium content (Yang et al. 2013, Vaudo et al. 2016) and can result in low growth rates and performance in bees (Tasei and Aupinel 2008, McAulay and Forrest 2019). Therefore, this pollen should be administered as part of a mixed pollen diet of diverse plants to provide bees with adequate nutrition for growth and reproduction. Mixing sunflower pollen with a wildflower pollen mix in a 1:1 ratio can maintain the medicinal effect without negatively affecting colony growth (Giacomini et al. 2021). Managers may also consider giving colonies access to sodium-enriched water if bees are not foraging outside (De Sousa et al. 2022). Our finding that sunflower is effective against Crithidia even after 5 yr in −20 °C storage is helpful information for those interested in using sunflower pollen to manage Crithidia infections in commercial bumble bee colonies. This research expands our understanding of sunflower pollen’s usefulness as a tool to improve bumble bee health.

Acknowledgments

We thank Koppert Biological Systems for bumble bee colonies. This work was supported by the National Institute of Food and Agriculture, US Department of Agriculture, the Center for Agriculture, Food and the Environment at the University of Massachusetts–Amherst, under USDA-AFRI-2018-08591 and USDA/CSREES (Multi-state Hatch) NE2001, as well as the National Science Foundation Graduate Research Fellowship. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA or NIFA.

Author Contributions

Alison Fowler (Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Writing – original draft [Equal]), Elisa Kola (Investigation [Equal], Writing – original draft [Equal]), and Lynn Adler (Conceptualization [Equal], Funding acquisition [Equal], Methodology [Equal], Project administration [Equal], Resources [Equal], Supervision [Equal], Writing – review & editing [Equal])

References