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Palmer-Young EC, Thursfield L. 2017. Pollen extracts and constituent sugars increase growth of a trypanosomatid parasite of bumble bees. PeerJ 5:e3297 https://doi.org/10.7717/peerj.3297 Pollen extracts increase growth of a trypanosome parasite of bumble bees
Evan C Palmer-Young Corresp. 1
1 Organismic and Evolutionary Biology, University of Massachusetts at Amherst, Amherst, MA, United States
Corresponding Author: Evan C Palmer-Young Email address: [email protected]
Phytochemicals produced by plants, including at flowers, function in protection against plant diseases, and have a long history of use against trypanosome infection. Floral nectar and pollen, the sole food sources for many species of insect pollinators, contain phytochemicals that have been shown to reduce trypanosome infection in bumble and honey bees when fed as isolated compounds. Nectar and pollen, however, consist of phytochemical mixtures, which can have greater antimicrobial activity than do single compounds. This study tested the hypothesis that pollen extracts would inhibit parasite growth.Extracts of six different pollens were tested for direct inhibitory activity against cell cultures of the bumble bee trypanosome gut parasite Crithidia bombi.
Surprisingly, pollen extracts increased parasite growth rather than inhibiting it. Experimental manipulations of growth media showed that supplemental monosaccharides (glucose and fructose) were sufficient to promote growth, while a common floral phytochemical (caffeic acid) with inhibitory activity against other trypanosomes had only weak inhibitory effects on Crithidia bombi. These results indicate that, although pollen is essential for bees and other pollinators, pollen may promote growth of intestinal parasites that are uninhibited by pollen phytochemicals and, as a result, can benefit from the nutrients that pollen provides.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2730v1 | CC BY 4.0 Open Access | rec: 16 Jan 2017, publ: 16 Jan 2017 1 Title Page
2 Title:
3 Pollen extracts increase growth of a trypanosome parasite of bumble bees
4 Short title/reference: Pollen extracts improve Crithidia growth
5 Authors:
6 Evan C Palmer-Young1
7 Affiliations:
8 1Organismic & Evolutionary Biology, University of Massachusetts Amherst, Amherst MA
9 01003
10 Corresponding author: Evan C Palmer-Young1
11 Email: [email protected]
12 13 Abstract
14 Phytochemicals produced by plants, including at flowers, function in protection against plant
15 diseases, and have a long history of use against trypanosome infection. Floral nectar and pollen,
16 the sole food sources for many species of insect pollinators, contain phytochemicals that have
17 been shown to reduce trypanosome infection in bumble and honey bees when fed as isolated
18 compounds. Nectar and pollen, however, consist of phytochemical mixtures, which can have
19 greater antimicrobial activity than do single compounds. This study tested the hypothesis that
20 pollen extracts would inhibit parasite growth. Extracts of six different pollens were tested for
21 direct inhibitory activity against cell cultures of the bumble bee trypanosome gut parasite
22 Crithidia bombi.
23 Surprisingly, pollen extracts increased parasite growth rather than inhibiting it. Experimental
24 manipulations of growth media showed that supplemental monosaccharides (glucose and
25 fructose) were sufficient to promote growth, while a common floral phytochemical (caffeic acid)
26 with inhibitory activity against other trypanosomes had only weak inhibitory effects on Crithidia
27 bombi. These results indicate that, although pollen is essential for bees and other pollinators,
28 pollen may promote growth of intestinal parasites that are uninhibited by pollen phytochemicals
29 and, as a result, can benefit from the nutrients that pollen provides.
30 31 Introduction
32 Plants provide nutrients that sustain the growth and reproduction of many animal species, but
33 also contain antimicrobial phytochemicals that may counteract infection in plant-eating animals
34 (de Roode et al., 2013). Insect pollinators such as bumble bees, whose diets consist exclusively
35 of phytochemical-rich nectar and pollen, are important for agricultural production. However, like
36 honey bees, bumble bees are threatened by parasite-related decline (Goulson et al., 2015).
37 Because bumble bees have abundant natural access to phytochemicals, antimicrobials from
38 flowers could provide a natural source of medicinal compounds that could counteract infection in
39 pollinator populations.
40 Trypanosomes are parasites that, in addition to afflicting over 12 million humans (Maslov
41 et al., 2013), can also be pervasive in populations of wild and managed bees (Schwarz et al.,
42 2015; Shykoff and Schmid-Hempel, 1991). With spread of parasites facilitated by use of shared
43 flowers (Durrer and Schmid-Hempel, 1994; Graystock et al., 2015), infection in some areas may
44 exceed 80% in bumble bees (Gillespie, 2010; Popp et al., 2012; Shykoff and Schmid-Hempel,
45 1991) and 90% in honey bees (Runckel et al., 2011). Correlative evidence implicates
46 trypanosome infection as a factor in honey bee colony loss. In Belgian honey bees, infection with
47 Lotmaria passim (formerly named and reported as Crithidia mellificae (Schwarz et al., 2015))
48 was correlated with colony death (Ravoet et al., 2013). In the United States, Lotmaria passim
49 infection intensity was over six-fold higher in hives that suffered from Colony Collapse Disorder
50 than in hives that did not collapse (Cornman et al., 2012). In bumble bees, Crithidia bombi
51 infection is similarly detrimental for individuals and colonies. Infection increased the rate of
52 death in starved workers (Brown et al., 2003), provoked potentially costly immune responses 53 (Sadd and Barribeau, 2013), altered foraging behavior and learning (Gegear et al., 2005), and
54 decreased colony fitness (Shykoff and Schmid-Hempel, 1991).
55 Phytochemicals have well-known antimicrobial properties that inhibit infection not only
56 in plants (Bennett and Wallsgrove, 1994; Huang et al., 2012), but also in animals that consume
57 phytochemical-rich plant materials (Karban and English-Loeb, 1997; de Roode et al., 2013;
58 Singer et al., 2009). Plant-based therapeutics have a long history of traditional use against
59 trypanosome infection, and recent studies have confirmed the antitrypanosomal activity of both
60 plant extracts and isolated phytochemicals (Merschjohann and Steverding, 2006; Santoro et al.,
61 2007a; Wink, 2012). Similarly, phytochemicals may have medicinal effects in bees infected with
62 the trypanosome C. bombi (Baracchi et al., 2015; Biller et al., 2015; Manson et al., 2010;
63 Richardson et al., 2015), such that nectar and pollen of phytochemical-rich wildflowers and
64 crops could provide medicinal resources for pollinators.
65 To date, all studies on the medicinal effects of phytochemicals on bees have tested single
66 compounds. However, plants contain mixtures of phytochemicals that can have synergistic
67 effects against both insects (Berenbaum and Neal, 1985; Berenbaum et al., 1991) and microbes
68 (Fewell and Roddick, 1993), including C. bombi (Palmer-Young et al., In revision). The
69 defensive compounds in phytochemical-rich plants, such as milkweed (Danaus spp.) (Gowler et
70 al., 2015), can also counteract pathogens of plant-eating insects and other animals (de Roode et
71 al., 2013). In a mouse model of Plasmodium falciparum malaria, crude Artemisia annua plant
72 extract had a stronger medicinal effect than did equivalent amounts of purified artemisinin
73 (Elfawal et al., 2012). Artemisia spp. extracts can also have inhibitory effects against
74 trypanosomes, such as the C. bombi relative Leishmania major, where the effects of crude plant 75 extracts and phytochemically complex essential oils can have greater antitrypanosomal activity
76 than do individual compounds (Efferth et al., 2011). Similarly, essential oil from Thymus
77 vulgaris plants was a more powerful inhibitor of Trypanosoma cruzi growth than was the
78 purified main constituent thymol (Santoro et al., 2007b). Together, these studies suggest that the
79 phytochemical mixtures found in natural plant materials may be more effective inhibitors of
80 parasites than are isolated chemicals.
81 Pollen and nectar consumed by bees contain diverse phytochemicals (Adler, 2001;
82 Dobson and Bergstrom, 2000; Jakubska et al., 2005). For example, over 100 compounds were
83 found in the nectar of a single orchid species (Jakubska et al., 2005), and bumble bees forage
84 from a variety of floral species throughout the growing season (Goulson and Darvill, 2004;
85 Heinrich, 2004; Vaudo et al., 2015). Pollen contains particularly high phytochemical
86 concentrations that can exceed those in nectar by several orders of magnitude (Detzel and Wink,
87 1993; London-Shafir et al., 2003; Palmer-Young et al., 2016). Hence, pollen could be expected
88 to have particularly strong effects on parasites that are susceptible to inhibition by
89 phytochemicals. However, studies in bees have found that, even though pollen consumption
90 increased expression of immune genes (Brunner et al., 2014), dietary pollen increased C. bombi
91 infection intensity (Conroy et al., 2016; Logan et al., 2005) in Bombus terrestris and B.
92 impatiens. One hypothesis to explain the positive effects of pollen was that nutrients in pollen
93 promote parasite growth. Pollen is rich in carbohydrates, proteins, and other nutrients that are
94 essential for bee reproduction (Roulston and Cane, 2000), but these nutrients could also benefit
95 parasites that can tolerate high phytochemical concentrations (Palmer-Young et al., 2016). 96 To test the alternative hypotheses that (a) pollen phytochemicals inhibit parasites and (b)
97 pollen nutrients benefit parasites, I tested the direct effects of six pollens, a known
98 antitrypanosomal nectar phytochemical, and monosaccharides on C. bombi growth in cell
99 culture.
100 Materials and Methods
101 Overview
102 Three experiments were conducted to elucidate the effects of pollen extracts on in vitro growth
103 of C. bombi cell cultures. These experiments evaluated the effects of (1) extracts of single
104 pollens, (2) extracts of mixed pollens, and (3) specific chemicals (sugar and the floral
105 phytochemical caffeic acid) in order to better understand the mechanisms by which pollen
106 extracts affected growth.
107 Parasite culturing
108 Crithidia bombi was isolated in 2013 by Ben Sadd from feces of wild Bombus impatiens near
109 Normal, IL by flow cytometry (Salathé et al., 2012) and kept frozen at -80 °C until several weeks
110 before the experiments. Thereafter, cells were grown at 27 °C in 50 cm2 tissue culture flasks and
111 transferred to fresh media every 3-4 days. No special permits were required for the collection.
112 Cultures are available upon request, provided appropriate documentation and permissions are
113 supplied. 114 Pollen and phytochemical treatments
115 Six types of bee-collected pollen—buckwheat (Fagopyrum esculentum), lotus (Nelumbo
116 nucifera), poppy (Papaver somniferum), rapeseed (Brassica napus), sunflower (Helianthus
117 annuus), and tea (Camellia sinensis)— were obtained from Changge Huading Wax Industry
118 (Henan, China) in 2015. The pollens were stored at -20 °C and sorted to remove heterogeneous
119 granules. For extraction, 6 g of pollen was incubated for 24 h at room temperature in constant
120 darkness with 20 mL of 50% aqueous methanol in a 50 mL conical tube. The 50% methanol was
121 used as a solvent due to its widespread application in phytochemical extraction of pollen (Serra
122 Bonvehi et al., 2001) and other plant tissues (Keinänen et al., 2001). Samples were shaken at 180
123 rpm on a shaker table for the first 20 min of the extraction. After 24 h, tubes were centrifuged
124 (30 min, 2700 g) and the supernatant removed, sterile-filtered, aliquoted, and stored at -80°C
125 until use. The mixed-pollen extract consisted of equal volumes of buckwheat, rapeseed, and
126 sunflower extracts, which were combined immediately before the experiment.
127 Caffeic acid was used as a representative phytochemical to evaluate possible negative and
128 positive effects of pollen constituents on C. bombi. This hydroxycinnamic acid is likely to be
129 widespread in bee diets, as it was the most widespread phytochemical in honey extracts, with
130 occurrence at detectable levels in all 14 tested types of Turkish honey (Can et al., 2015);
131 cinnamic acids and other phenolics are also abundant in pollen (Almaraz-Abarca et al., 2004;
132 Campos et al., 1997; Serra Bonvehi et al., 2001). Caffeic acid has strong antitrypanosomal
133 effects on Leishmania donovani, Trypanosoma cruzi, and T. brucei (Grecco et al., 2014;
134 Tasdemir et al., 2006), which suggested that caffeic acid could inhibit C. bombi as well.
135 However, caffeic acid is also a powerful antioxidant, with ability to scavenge reactive oxygen 136 species that exceeds that of ascorbic acid and is comparable to that of tocopherols (Almaraz-
137 Abarca et al., 2004; Chen and Ho, 1997). Antioxidant activity of caffeic acid and other pollen
138 components (Almaraz-Abarca et al., 2004) might protect Crithidia bombi from stress incurred
139 during the experiment, such as shaking and handling, and in the wild, where parasites encounter
140 temperature changes, osmotic shock, UV light, and pro-oxidant enzymes of the bee immune
141 system that may contribute to oxidative stress (Sadd and Barribeau, 2013; Vanaerschot et al.,
142 2014). For experiments, caffeic acid was dissolved to a concentration of 4 mg mL-1 in 50%
143 methanol and tested at a final concentration of up to 333 ppm. This concentration is more than
144 30-fold the levels that occur in most types of honey (Can et al., 2015) and 10-fold the mean
145 cinnamic acid concentration in pollen (Serra Bonvehi et al., 2001). Therefore, the tested
146 concentration range was likely to detect any antitrypanosomal effects that could be attributed to
147 phytochemical consumption by bees.
148 The addition of sugar to the medium was also tested for positive or negative effects on
149 growth. Bee-collected pollen, such as that used to create the extracts in this study, is rich in
150 sugars from nectar, which are added to the pollen by bees during collection (Roulston and Cane,
151 2000). Previous experiments with C. bombi cell cultures showed that growth was strongly
152 inhibited by addition of 20% sugar to growth medium (Cisarovsky and Schmid-Hempel, 2014).
153 However, other trypanosomes such as T. brucei prefer sugars to proteins as a carbon source
154 (Lamour et al., 2005), which suggests that addition of sugar to the peptone- and liver-based
155 growth medium (Salathé et al., 2012) could enhance growth. The sugar solution consisted of
156 20% w/v (10% dextrose (d-glucose) + 10% fructose) in 50% methanol. This sugar concentration
157 was chosen to slightly exceed the likely sugar concentration in the pollen extracts, which was
158 estimated as ~10%. This estimate was based on a sugar content of ~30-40% by weight in the 159 pollen (Campos et al., 2008; Herbert and Shimanuki, 1978; Roulston and Cane, 2000; Todd and
160 Bretherick, 1942), with ~30% pollen in the extract. The sugar composition was chosen to reflect
161 the roughly equal amounts of dextrose and fructose that have been found in nectar and honey
162 (London-Shafir et al., 2003; Ohmenhaeuser et al., 2013). Although pollen contains sugars other
163 than monosaccharides (Herbert and Shimanuki, 1978), monosaccharides were used because the
164 bee intestine rapidly hydrolyzes disaccharides to glucose and fructose (Nicolson, 1998), which
165 likely leaves only monosaccharides in the distal intestine where trypanosomes become
166 established (Lipa and Triggiani, 1988; Schwarz et al., 2015). The sugar solution was tested at up
167 to 8.3% concentration by volume (16.7 g L-1 monosaccharides in growth medium).
168 Experimental design
169 Each experiment tested the effects of treatments on growth of parasite cell cultures in 96-well
170 microplates. The first experiment tested extracts of six different species of pollen. The second
171 experiment tested the effects of buckwheat, rapeseed, and sunflower pollen extracts, individually
172 and in a mixture that consisted of equal proportions of each of the three extracts (i.e., one-third
173 buckwheat, one-third rapeseed, and one-third sunflower extract by volume). The third
174 experiment tested the effects of added chemicals, which included the common floral
175 phytochemical caffeic acid and a sugar solution. This third experiment included buckwheat
176 pollen extract as a positive control to verify the effects of pollen extracts observed in the
177 previous two experiments.
178 To test the effects of pollen extracts, extract of each of the six pollens was dissolved at
179 six concentrations by two-fold serial dilution. Concentrations ranged from 0-5% (for pollen
180 extracts) or 0-8.3% (for chemical additions) final concentration by volume in growth medium. 181 Additional 50% methanol was added to samples of lesser concentrations to equalize methanol
182 concentrations across samples. The inner 48 wells of a 96-well plate were filled with 100 µL of
183 treatment solution (at 2x final concentration) and 100 µL of a suspension of C. bombi cells (103
184 cells µL-1), to achieve an initial cell density of 500 cells µL-1 (250 cells µL-1 for chemical
185 addition experiment). Outer wells were filled with 200 µL sterile water to mitigate edge effects.
186 Plates were sealed with laboratory film and placed inside zippered sandwich bags to minimize
187 evaporation. Samples were incubated without shaking at 27 °C in a dark incubator. Growth was
188 measured as optical density (630 nm) at 24 h intervals for 5 d by a spectrophotometer. Before
189 each growth measurement, plates were shaken on a microplate shaker (30 s, 1000 rpm, 4 mm
190 orbit) to homogenize and resuspend the cells. In addition, immediately before each
191 spectrophotometer reading, the plate cover used during incubation was exchanged for a dry plate
192 cover under sterile conditions, to prevent condensation from interfering with optical density
193 measurements. Wells that contained treatment media without cells and were used to control for
194 changes in optical density independent of parasite growth. Experiments included n = 8 (for
195 pollen extracts) or n = 5 (for chemical additions) replicate samples per treatment concentration,
196 plus the n = 2 negative control wells of cell-free treatment medium.
197 Analysis
198 Linear regression was used to test for concentration-dependent changes in parasite growth.
199 Treatment concentration was used as the predictor variable. Growth integral (i.e., area under the
200 curve of growth vs. time, estimated using a model-free spline (Kahm et al., 2010)) was used as
201 the response variable. Separate models were fitted for each pollen extract or chemical. P-values 202 were adjusted with a Bonferroni correction to account for multiple tests within each experiment.
203 Graphs were produced with the R package ggplot2 (Wickham, 2009).
204
205 Results
206 Extracts of each of the six pollens resulted in increased C. bombi growth, as measured by the 5-
207 day growth integral (Figure 1); the increase in growth was significant in analysis by linear
208 regression (Table 1). Relative to the pollen-free control, addition of 5% extract of each pollen
209 resulted in approximately 50% more growth over 5 d. Effects were similar for a mixture of
210 buckwheat, rape, and sunflower extracts (Figure 2).
211 In the test of specific additional chemicals, both the buckwheat pollen extract (positive
212 control) and 20% sugar solution resulted in increased growth (Figure 3, Table 1), whereas the
213 common floral phytochemical caffeic acid (4 * 103 ppm stock solution) had only weakly
214 inhibitory effects at up to 333 ppm, which is an order of magnitude higher than any
215 concentration documented among different types of honey (Can et al., 2015). Although
216 inhibition was statistically significant (Table 1), the highest concentration resulted in <50%
217 growth inhibition, which precluded estimation of a standard dose-response curve to determine
218 the EC50 concentration.
219 220 Discussion
221 These experiments indicate that pollen extracts can increase growth of an intestinal parasite, and
222 that the growth-promoting effects of pollen extracts can be reproduced by addition of similar
223 amounts of a sugar solution. Pollen phytochemicals appear to be insufficient to stop growth of C.
224 bombi, and moreover, pollen appears to contain substances that improve trypanosome growth.
225 This result is consistent with previous experiments that showed a decrease in infection intensity
226 in bees deprived of pollen, and the results presented here suggest a mechanism by which pollen
227 may promote trypanosome infection. The positive effects of pollen nutrients on C. bombi, a
228 hindgut trypanosome, suggests the potential for facilitation of nutrient-limited hindgut parasites
229 by midgut parasites that interfere with nutrient absorption. In addition, the phytochemical
230 tolerance of C. bombi relative to that of bloodstream trypanosomes invites further study on
231 adaptation to phytochemicals in different trypanosome species.
232 Phytochemical insensitivity
233 Crithidia bombi growth was not inhibited by any of the pollen extracts. This was unexpected in
234 the context of the current literature on bumble bee-Crithidia interactions, which has suggested
235 that phytochemical ingestion can counteract proliferation of C. bombi (Baracchi et al., 2015;
236 Manson et al., 2010; Richardson et al., 2015). On the contrary, growth was actually increased in
237 the presence of the extracts. Similarly, growth of C. bombi in the present study was only weakly
238 inhibited (<50% decrease in growth integral, Figure 3) by caffeic acid at concentrations of over
239 300 ppm. This concentration is far higher than the levels that occur naturally in honey and pollen
240 (Serra Bonvehi et al., 2001); for example, among 14 types of honey, none contained >27 ppm
241 (Can et al., 2015). Insensitivity of C. bombi to hydroxycinnamic acids is consistent with previous 242 results (Palmer-Young et al., 2016), and striking in light of work on other trypanosomes. For
243 comparison, concentrations needed for 50% growth inhibition (“EC50”) of L. donovani, T.
244 brucei, and T. cruzi ranged from 1.1-56 ppm caffeic acid (Grecco et al., 2014; Tasdemir et al.,
245 2006), and
246 C. bombi appears to be well adapted to phytochemicals, including those that are toxic to
247 other trypanosomes and even those initially shown to reduce infection intensity. For example, C.
248 bombi exhibited EC50 values for several phenolics that were orders of magnitude higher than
249 those reported for other trypanosome species (Palmer-Young et al., 2016), and was robust to
250 thymol, anabasine, and nicotine under controlled conditions (Biller et al., 2015; Thorburn et al.,
251 2015). Although the present study did not address possible host-mediated effects of
252 phytochemicals on infection, such as phytochemical-induced stimulation of immune responses
253 (Borchers et al., 1997; Mao et al., 2013) or change in gut kinetics (Tadmor-Melamed et al., 2004)
254 that have been observed in other species and could alter trypanosome attachment to the gut wall
255 (Schwarz et al., 2015), the fact that none of the six pollens showed inhibitory activity
256 demonstrates that C. bombi is robust to many of the phytochemicals in the diet of its hosts.
257 Many trypanosomes complete their life cycle in two hosts, which may include insects,
258 mammals, and plants (Maslov et al., 2013). It would be intriguing to use the comparative method
259 to test whether evolutionary history is predictive of phytochemical tolerance. To accomplish this,
260 future studies could compare the phytochemical tolerance of species that occupy niches with
261 different levels of phytochemical exposure. In order of decreasing intensity of phytochemical
262 exposure, these could include (a) species that utilize plants as hosts, (b) those that are gut
263 parasites of herbivores, and (c) species and life stages that live in the blood and are transmitted 264 by blood-feeding insects. Trypanosomes with an evolutionary history of phytochemical exposure
265 would be expected to have higher phytochemical tolerance than those that encounter
266 phytochemicals only occasionally or indirectly.
267 Increase in growth was reproduced by addition of sugar
268 The growth-promoting properties of the pollen extracts may be attributable to its constituent
269 nutrients, in particular monosaccharides. Pollen collected by corbiculate bees, such as bumble
270 bees, is moistened with nectar, which renders it sufficiently sticky to be carried back to the hive
271 in the bee’s corbicula (pollen basket) (Roulston and Cane, 2000). As a result, bee-collected
272 pollen contains considerable amounts of carbohydrate, including up to 40% sugars by weight
273 (Roulston and Cane, 2000; Todd and Bretherick, 1942). Although the osmolarity of very high
274 (~20%) sugar concentrations can kill C. bombi as well as other microbes (Cisarovsky and
275 Schmid-Hempel, 2014), the effects found in this study indicate that addition of sugar at low
276 concentrations is beneficial for trypanosome growth.
277 The monosaccharides added to the growth medium would have increased the sugar
278 content several-fold, providing up to 16.7 additional g L-1 monosaccharides in addition to the 2.2
279 g L-1 in the original growth medium (1.8 g L-1 from fructose + 0.4 g L-1 from dextrose in liver
280 broth (Salathé et al., 2012)). This additional sugar may have increased the quality of the media
281 for C. bombi energy production. Trypanosomes rely on glycolysis for energy production (Mazet
282 et al., 2013). Although they are capable of using peptides for energy (Bringaud et al., 2006),
283 peptide metabolism is dramatically reduced in the presence of glucose, which suggests that
284 glucose is a preferred energy source (Lamour et al., 2005). The trypanosomes that infect bees,
285 which consume carbohydrate-rich diets, may be particularly adapted to utilization of 286 carbohydrates. In a genomic comparison between Leishmania major and the honey bee gut
287 parasite Lotmaria passim (n.b. Originally reported as C. mellificae), genes related to
288 carbohydrate metabolism were enriched in the bee parasite compared to its bloodborne relative
289 (Runckel et al., 2014). Genomic studies may reveal whether carbohydrate metabolism is also
290 well developed in C. bombi, which is a close relative of L. passim (Schwarz et al., 2015).
291 The stimulatory effect of sugar on C. bombi growth raises the question of possible
292 facilitation of hindgut trypanosome infections by co-occurring infections that impair nutrient
293 absorption, such as Nosema ceranae and Nosema apis. Nosema spp. have been implicated in
294 collapse of honey bee colonies (Cornman et al., 2012; Higes et al., 2009) and may infect bumble
295 bees as well (Graystock et al., 2013). Field studies have found positive correlations between
296 Nosema apis and trypanosome infections in honey bees (Cornman et al., 2012), which provides
297 suggestive evidence for positive effects of Nosema spp. on gut trypanosomes. The present study
298 suggests a mechanism by which Nosema infection could contribute to trypanosome infection via
299 negative effects on sugar absorption in bees. Healthy bees and other nectivorous insects have an
300 excellent ability to digest and absorb sugars from nectar (Nicolson, 1998), which may explain
301 why there was no effect of dietary sugar concentration on infection intensity in B. impatiens
302 (Conroy et al., 2016) . However, gut infection by microsporidians can disrupt the midgut
303 epithelium (Higes et al., 2008). Injury to midgut tissue may decrease absorption of sugar in
304 hosts, as suggested by the decreased hemolymph sugar concentrations and increased hunger
305 observed in Nosema-infected bees (Mayack and Naug, 2009). As a result of Nosema-induced
306 malabsorption, more sugar may reach the hindgut, and thereby increase the supply growth-
307 limiting carbohydrate to trypanosomes. To test the hypothesis that Nosema-related 308 malabsorption facilitates infection by trypanosomes, future experiments could test the effects of
309 microsporidian infection on fecal carbohydrate content and trypanosome infection intensity.
310 Pollen in pollinator communities
311 Pollen, like nectar, is an indispensable source of nutrients for bees and other pollinators that
312 supports insect immunity (Brunner et al., 2014), survival (Conroy et al., 2016), and reproduction
313 (Vaudo et al., 2015). However, pollen may nourish parasites as well as hosts. Although
314 phytochemicals may reduce growth of some microbes, parasites of phytophagous animals are
315 likely adapted to the phytochemicals and concentrations found in the diets of their hosts.
316 Moreover, in coevolved obligate parasites such as Crithidia and other trypanosomes (Maslov et
317 al., 2013), there may be substantial overlap in the nutrient requirements of parasites and hosts.
318 Shared nutritional requirements, and the increased fitness of parasites in well-nourished hosts,
319 may result in tradeoffs between starvation of parasites and starvation of hosts, and may explain
320 the utility of anorexia as a defense against parasites in some taxa (Parker et al., 2011). The results
321 found here exemplify the trade-offs between host health and defense, underline the difficulty of
322 eradicating well-adapted parasites without compromising host fitness, and suggest that natural
323 selection may act across all levels of tritrophic interactions.
324 Acknowledgements
325 Thanks to Lynn Adler, Jonathan Giacomini, and Rebecca Irwin for inspiring the study; to Ben
326 Sadd for providing the parasite cell culture; to Rob Wick, William Manning, and Jeffrey
327 Blanchard for sharing laboratory space and equipment; to Anastasiya Mirzayeva for technical
328 assistance; to Matthias Kahm, Hadley Wickham, and the R Core team for statistical resources; 329 and to the reviewers for their critique of the manuscript. Funders are gratefully acknowledged in
330 the separate funding statement.
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534 Table 1(on next page)
Effects of pollen extracts and supplemental chemicals on growth.
Estimates and p-values are for linear regression after Bonferroni correction for multiple testing within each experiment.
*Mix treatment consisted of equal proportions of buckwheat, rape, and sunflower extracts. 1 Table 1. Effects of pollen extracts and supplemental chemicals on growth. Estimates and p-values are 2 for linear regression after Bonferroni correction for multiple testing within each experiment.
A. Single pollens Treatment Slope (β) Std. Error T p (T) Buckwheat 2.60 0.21 12.52 <0.001 Lotus 1.88 0.16 11.52 <0.001 Poppy 2.44 0.31 7.91 <0.001 Rape 1.95 0.22 8.69 <0.001 Sunflower 1.58 0.13 11.70 <0.001 Tea 2.42 0.24 10.15 <0.001 B. Mixed Pollens Buckwheat 1.90 0.18 10.63 <0.001 Rape 2.09 0.17 12.12 <0.001 Sunflower 1.90 0.16 12.06 <0.001 Mix* 1.38 0.11 12.36 <0.001 C. Chemical additions Buckwheat 1.39 0.14 10.02 <0.001 Caffeic acid -0.43 0.17 -2.55 0.050 Sugar 1.56 0.14 11.11 <0.001
3 *Mix treatment consisted of equal proportions of buckwheat, rape, and sunflower extracts. Figure 1(on next page)
Individual pollen extracts increased parasite growth.
Extracts of six types of pollen were tested at up to 5% concentration. Each panel shows the 5 d growth integral (i.e., area under the curve of OD vs. time) for parasites exposed to 50% methanol extracts of one of the six types of pollen. Additional 50% methanol was added to samples of lesser concentrations to equalize methanol concentrations across samples. Points and error bars show means and standard errors for each concentration (n = 8). OD: optical density (630 nm). See Supplementary Figures 1-3 for complete growth curves. Buckwheat Lotus Poppy
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10 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Concentration (% extract) Figure 2(on next page)
Mixed and individual pollen extracts each increased growth.
The treatments consisted of extracts of individual pollens and a mixture (“mix”) of equal proportions of buckwheat, rapeseed, and sunflower pollen. Each panel represents a different pollen extract. Additional 50% methanol was added to samples of lesser concentrations to equalize methanol concentrations across samples. Points and error bars show means and standard errors for each concentration (n = 8). Buckwheat Rape
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10 0 1 2 3 4 5 0 1 2 3 4 5 Concentration (% extract) Figure 3(on next page)
An antitrypanosomal floral phytochemical had weak effects on growth, whereas supplemental sugar increased growth.
Each panel shows the growth curve for parasites exposed to one of the chemical treatments. Buckwheat pollen extract was used as a positive control to confirm increased growth in the presence of pollen extract. The caffeic acid solution contained 4 * 10 3 ppm caffeic acid in 50% methanol. The sugar solution consisted of 20% w/v monosaccharides (10% dextrose + 10% fructose in 50% methanol). Chemical solutions and pollen extract were added at the same final concentrations (up to 8.3% in prepared samples), which corresponds to up to 333 ppm caffeic acid and up to 16.7 mg L-1 additional sugar. Additional 50% methanol was added to samples of lesser concentrations to equalize methanol concentrations across samples. Points and error bars show means and standard errors for each concentration (n = 5). ● Sugar (20% w/v) 30
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Caffeic acid (4000 ppm)
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0.0 2.5 5.0 7.5 Concentration (percent by volume)