DOI: 10.5061/dryad.dv41ns1v9
title: Assessing chemical mechanisms underlying the effects of sunflower pollen on a gut pathogen in bumble bees

description: Many pollinator species are declining due to a variety of interacting
 stressors including pathogens, sparking interest in understanding factors
 that could mitigate these outcomes. Diet can affect host-pathogen
 interactions by changing nutritional reserves or providing bioactive
 secondary chemicals. Recent work found that sunflower pollen (Helianthus
 annuus) dramatically reduced cell counts of the gut pathogen Crithidia
 bombi in bumble bee workers (Bombus impatiens), but the mechanism
 underlying this effect is unknown. Here we analyzed methanolic extracts of
 sunflower pollen by LC-MS and identified triscoumaroyl spermidines as the
 major secondary metabolite components, along with a flavonoid
 quercetin-3-O-hexoside and a quercetin-3-O-(6-O-malonyl)-hexoside. We then
 tested the effect of triscoumaroyl spermidine and rutin (as a proxy for
 quercetin glycosides) on Crithidia infection in B. impatiens, compared to
 buckwheat pollen (Fagopyrum esculentum) as a negative control and
 sunflower pollen as a positive control. In addition, we tested the effect
 of nine fatty acids from sunflower pollen individually and in combination
 using similar methods. Although sunflower pollen consistently reduced
 Crithidia relative to control pollen, none of the compounds we tested had
 significant effects. In addition, diet treatments did not affect
 mortality, or sucrose or pollen consumption. Thus, the mechanisms
 underlying the medicinal effect of sunflower are still unknown; future
 work could use bioactivity-guided fractionation to more efficiently target
 compounds of interest, and explore non-chemical mechanisms. Ultimately,
 identifying the mechanism underlying the effect of sunflower pollen on
 pathogens will open up new avenues for managing bee health.
description: The general approach was similar for all assays and followed
 previously published protocols (Giacomini et al. 2018; LoCascio et al.
 2019a; Richardson et al. 2015). Commercial experimental colonies (BioBest,
 Leamington, Canada) were confirmed to be <i>Crithidia</i>-free
 by biweekly subsampling, and the <i>Crithidia</i> strain used
 in these assays was originally collected in 2015 from infected wild
 <i>B. impatiens</i> at a farm in Hadley, Massachusetts USA
 (42°21'51.93"N, 72°33'55.88"W) and maintained in
 commercial colonies thereafter. All colonies were fed mixed wildflower
 pollen (Koppert Biological Systems, Howell, Michigan, USA or CC Pollen
 Inc., Phoenix, Arizona, USA). Worker bees were inoculated after a 1-2 hr
 starving period with either 10 µl (secondary compounds) or 15 µl (fatty
 acids) of a 25% sucrose solution containing 600 cells/µl, well within the
 range of natural fecal <i>Crithidia</i> concentrations
 (Otterstatter and Thomson 2006). Upon inoculation, bees were placed in
 individual 18.5 ml vials (secondary compounds) or Placon 473 ml cups
 (fatty acids) and assigned to diet treatments that they received for 7
 days along with 30% sucrose solution that was replaced daily (secondary
 compounds) or every other day (fatty acids). Bees were maintained in
 darkness at 27<sup>o</sup>C in an incubator during assays.
 After 7 days, bees were dissected to remove the gut, which was ground in
 300 µl of ¼ strength Ringer’s solution (Sigma-Aldrich – Fluka 96724).
 After 4 hr, a 10 µl subsample was removed and moving
 <i>Crithidia</i> cells were counted on a 0.02 µl field of
 vision on the grid of a hemacytometer.      For the secondary compound assays
 we used callows, which are bees that had emerged from pupae within the
 last 24 hr. We isolated pupal clumps from colonies, collected callows as
 they emerged and fed them wildflower pollen for two days before
 inoculating them to enter the experiment. Because callows emerged without
 nestmates, this was an extra precaution to ensure they were free of
 <i>Crithidia</i> (which infects adults), but these bees also
 did not receive gut microbiota from their nestmates that could influence
 interactions with <i>Crithidia</i> (Koch and Schmid-Hempel
 2011). For the fatty acid assays, we pulled adult workers directly from
 colonies and inoculated them to enter the experiment. This method does not
 control for worker age, but allows bees to acquire their colony’s gut
 microbiome. Within each assay, bees with pollen treatments were compared
 to controls under the same conditions, so while our methods varied, we can
 still assess diet effects within each experiment. We also included a
 measure of bee size for all assays since smaller bees often have higher
 infections (e. g., Richardson et al. 2015). For the secondary compound
 assays we used callow mass, and for fatty acids we used radial cell
 length, which is highly correlated with other measures of body size
 (Nooten and Rehan 2020). In the triscoumaryl spermidine experiment, we
 measured both callow mass and radial cell length and they were highly
 correlated (Pearson’s r = 0.85, n = 259).  For all assays we recorded daily
 mortality, and measured daily pollen and sucrose consumption once per bee,
 except for omitting sucrose consumption in the triscoumaroyl spermidine
 assay. Pollen and sucrose rations were typically weighed upon providing
 them to bees and then approximately 24 hr later (48 hr in the first fatty
 acid assay).   <i>Secondary
 Chemical Bioassays</i>. We tested the effects of two major secondary
 compounds found in sunflower pollen, a triscoumaroyl spermidine and rutin,
 at four concentrations each. We used rutin as a proxy for the quercetin
 glycosides identified in sunflower pollen because rutin is also a
 quercetin glycoside and available commercially. Quercetin glycosides are
 reported to show equivalent activity across a range of analogues against
 microorganisms including trypanosomes (da Silva et al. 2019; Marin et al.
 2017), suggesting that rutin provides a suitable proxy for other quercetin
 glycosides. Tri-<i>p</i>-coumaroyl
 spermidine (‘triscoumaryl spermidine’ hereafter) was synthesized in the
 laboratory as described in Online Resource 1, while rutin was purchased
 commercially (Sigma-Aldrich, R5143-50G). Both were added to buckwheat
 pollen at 1% (very high), 0.1% (high), 0.01% (medium), or 0.001% (low)
 concentrations wt/wt. For context, we found 12 mg/g of triscoumaroyl
 spermidine in pollen (1.2%; comparable with the ‘very high’ treatment) and
 0.2% quercetin glycosides in pollen, similar to the ‘high’ level. Thus,
 our concentrations are within natural ranges. Compounds were mixed with
 buckwheat pollen and compared to pure buckwheat pollen as a negative
 control and sunflower pollen as a positive control (sunflower and
 buckwheat pollen came from Changge Huading Wax Industry Co., Ltd., Henan,
 China; except that buckwheat came from Fuyang Import and Export Ltd for
 the final fatty acid assay). Compounds were added to dry pollen, which was
 then mixed with distilled water for all treatments to create a paste that
 was frozen at -20<sup>o</sup>C until use. Details of mixing
 secondary compounds with pollen and preparing pollen balls are in Online
 Resource 2. The
 triscoumaryl spermidine assay was conducted from July 17 through August
 19, 2015, using 108 bees from three experimental colonies, ultimately
 including 15-20 bees/treatment. The rutin experiment was conducted from
 November 3, 2015 through April 7, 2016 and used 292 bees from 12
 experimental colonies, ultimately including 45-54
 bees/treatment.   <i>Fatty
 Acid Bioassays</i>. We tested the effects of several fatty acids
 singly and in combination. We chose the most common ones detected in
 sunflower pollen that were commercially available, and tested each at
 typical concentrations based on published literature (Nicolson and Human
 2013; Yang et al. 2013).  We conducted three assays
 that each tested individual or combinations of fatty acids added directly
 to control pollen as powders or liquids. Compounds tested in the first
 assay were linoleic (1.67 mg/g; CAS 60-33-3), lauric (12.39 mg/g; CAS
 143-07-7), caprylic (1.46 mg/g; CAS<b> </b>124-07-2), palmitic
 (8.46 mg/g; CAS 57-10-3), and decanoic (0.14 mg/g; CA: 334-48-5) acids.
 The second assay included linolenic (7.61 mg/g; CAS 463-40-1), myristic
 (1.62 mg/g; CAS 544-63-8), stearic (0.65 mg/g; CAS 57-11-4) and oleic
 (5.39 mg/g; CAS 112-80-1) acids. The third assay tested all nine fatty
 acids combined in a single treatment (using the same concentrations as in
 individual assays) and sunflower oil, which contains a mix of fatty acids
 (37.07 mg/g; Organic 365 Everyday Value Products, Whole Foods Market,
 Austin, Texas, USA), to assess whether fatty acids had interactive effects
 that would not be detected in assays of individual compounds. Details of
 suppliers and concentration calculations are provided in Online Resource
 2. Fatty acids were added to dry pollen with a coffee grinder, and then
 distilled water was added in a 6:1 or 7:1 (third assay only) ratio to make
 a paste. As with the secondary chemical assays, compounds were added to
 buckwheat pollen and compared to buckwheat and sunflower pollen as
 negative and positive controls, respectively. However, in the second assay
 we did not have sufficient buckwheat pollen and instead added fatty acids
 to mixed wildflower pollen (CC Pollen Inc. Phoenix, Arizona, USA) and used
 wildflower pollen as the negative control.
 <b>            </b>We conducted the first assay from
 September 28, 2018 to February 6, 2019 using 246 bees from 14 experimental
 colonies, including 32-37 bees per fatty acid treatments and 47 in the
 buckwheat control. The second was conducted from February 20 to April 12,
 2019, using 167 bees from 5 experimental colonies, including 25-31
 bees/treatment. The third assay was conducted from July 18 to 26, 2019
 using 106 bees from 4 experimental colonies, including 25-27
 bees/treatment.
description: All variables for all CSV files are described in the
 'ReadMe' text file. A single R script was
 used to analyze all data and is also uploaded.  There
 are missing values in the <em>Crithidia </em>count datasets
 when bees died before the 7-day span of the bioassays.