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The presence of a larval honey bee parasite, Ascosphaera apis, on flowers reduces pollinator visitation to several plant species
Acta Oecologica 96 (2019) 49–55
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The presence of a larval honey bee parasite, Ascosphaera apis, on flowers reduces pollinator visitation to several plant species
T
Babak Yousefi, Bertrand Fouks∗ Department of Biology, University of North Carolina at Greensboro, 321 McIver Street, Greensboro, NC, 27412, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Plant-pollinator interactions Behavioral immunity Avoidance behavior Host-parasite interactions Tripartite interactions Floral evolution
Many floral traits have evolved through pollinator-mediated selection. In selecting flowers for forage, pollinators rely on multi-modal flower signals and innate preferences. However, pollinators are known for their flexible foraging behavior which varies with experience and numerous cues, such as the presence of competitors and floral resource availability. The shared use of flowers by many pollinators leads to high rate of parasite transmission among pollinators, and flowers can be seen as hot-spots of parasite dispersal. Throughout the animal kingdom, behavioral adaptations enable avoidance of parasite exposure. Bumblebees are known to avoid contaminated flowers, however nothing is known regarding the impact of parasite presence on overall pollinator visitation. In this study, we measured the effect of a larval honey bee parasite, the fungus Ascosphaera apis causing chalkbrood disease, on hymenopteran pollinator visitation of several plant species in a natural setting. Several contaminated flowering plant species suffered not only the subsequent reduction of visitation by honey bees but also the subsequent reduction of their overall pollinator visitation. Two plant species with similar flower architecture were the least impacted by the presence of the spores. This study highlights the negative impact of the presence of pollinator parasites for plants and its possible consequences for floral evolution.
1. Introduction Angiosperms have evolved interactions with animals to facilitate their reproduction (Dilcher, 2000). Pollinator-mediated selection resulting from those interactions is a mechanism underlying floral diversity (Fenster et al., 2004; Sapir and Armbruster, 2011). In theory, pollinator-mediated selection favors floral traits that maximize reproductive fitness via pollen export and import (Morgan, 1992). Thus many floral traits have evolved to attract pollinators (Fenster et al., 2004; Schiestl and Johnson, 2013) and pollinators rely on innate preferences (Goulson et al., 2007) and associative learning to forage on flowers (Chittka and Thomson, 2001). Moreover, pollinators rely on multi-modal signals, such floral color and scent, to choose flowers (Leonard et al., 2011a; Leonard and Masek, 2014). Pollinators also exhibit flexibility in their foraging behavior as they gain experience (Riffell et al., 2014). This leads to complex decision making and behaviors of pollinators relying on multi-modal signals to choose flowers to forage (Chittka and Thomson, 2001; Leonard et al., 2011b), in order to optimize their exploitation of floral resources. Parasites are of major ecological and evolutionary importance (Hatcher and Dunn, 2011), and have been observed influencing diet
choices in many host species (Hart, 2011; de Roode and Lefèvre, 2012; Curtis, 2014). Parasites and related diseases represent a significant threat, reducing organismal reproduction and lifespan (Bonsall, 2004), and acting as a strong evolutionary force (Bell and Maynard Smith, 1987). Moreover, parasites can indirectly impact several aspects of ecological relationships (Wood and Johnson, 2015), including trophic (Lafferty et al., 2006, 2008), competitive (Hatcher et al., 2006) and mutualistic interactions (Liere and Larsen, 2010; Wood et al., 2018). Parasite threat is inherently dependent on the likelihood of parasite encounter and transmission. Shared use of resources plays a major role in the dynamics of parasite dispersal (Prüss-Ustün et al., 2002). Since parasites often populate environments in which they encounter numerous species, non-host species can also be important in parasite dispersal (Rigaud et al., 2010; Dunn and Perkins, 2012). Parasites transmitted among species can be dispersed widely and to novel locations (Tatem et al., 2006). Flowers have been shown to play a role in parasite transmission among pollinator species, with flowers acting as hot-spots of disease dispersal (Durrer and Schmid-Hempel, 1998; Graystock et al., 2015). While some plant-pollinator systems are specialized, the vast majority of flowering plant species are visited by multiple pollinator species in a complex web of interactions (Waser
∗ Corresponding author. Present address: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, P. O. Box 1066 Blindern, NO-0316, Oslo, Norway. E-mail address: [email protected] (B. Fouks).
https://doi.org/10.1016/j.actao.2019.03.006 Received 6 December 2018; Received in revised form 26 February 2019; Accepted 18 March 2019 1146-609X/ © 2019 Elsevier Masson SAS. All rights reserved.
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1 ml of distilled water and the number of spores was counted at a 10−3 dilution in a phase hematocytometer (Hausser Scientific®, Horsham, PA, USA). Spore solutions were combined to obtain a concentration of 2 × 106 spores/ml (for a total of 20 ml). Spore solutions were either made immediately before observations or were stored at 6 °C in the dark until use (max. 4 days).
et al., 1996; Fontaine et al., 2006). The vectoring of parasites by nonhost pollinator species during shared flower use may be of great importance in pollinator–parasite interactions (Evison et al., 2012; McArt et al., 2014), and there is increasing evidence for the incidence and impact of parasite transmission among pollinator taxa (Evison et al., 2012; Fürst et al., 2014; McMahon et al., 2015; Bailes et al., 2018). The selective pressure parasites put on their hosts drives the adaptation of a wide range of defense mechanisms (Schmid-Hempel, 2011). One of these mechanisms is the adaptation of host behavior, with behavioral avoidance (e.g. physical avoidance) being the most important and wide-ranging strategy in the animal kingdom (Hart, 2011). Behavioral avoidance is the first line of defense against infection, as it can prevent or reduce parasite encounter (Schmid-Hempel, 2011), and is thus likely to be a highly cost-effective form of defense. Few studies demonstrate the impact of parasites on foraging pollinator decisionmaking and flower choice (Fouks and Lattorff, 2011; Richardson et al., 2016). Behavioral avoidance of flowers contaminated by parasites was first observed in bumble bees (Fouks and Lattorff, 2011). Bumble bees reduced their number of visits and feeding duration on contaminated flowers (Fouks and Lattorff, 2011, 2013). Avoidance of parasite-infested flowers may negatively impact plant fitness; as reduced pollinator visitation can lead to pollen limitation (Knight et al., 2005; Harder and Aizen, 2010) and decreased plant reproductive success (Irwin and Brody, 1998, 1999; Sahli and Conner, 2006, 2007). The fungus Ascosphaera apis is a common parasite of honey bees, provoking chalkbrood disease and killing larvae (Evison and Jensen, 2018). Evidence suggests A. apis may be transmitted horizontally through large number of pollinator species via the shared use of flowers (Evison et al., 2012; Mc Art et al., 2014). While A. apis is specific to honey bee brood, it is also known of cross-species transmission to the brood of some wild bees (i.e. Xylocopa californica and Megachile rotundata), and the adult stage of bumble bees (Maxfield-Taylor et al., 2015; Evison and Jensen, 2018). Furthermore, honey bees (2 individuals out of 12), bumble bees (34 out of 114), solitary bees (50 out of 56), wasps (27 out of 53) and hover-flies (26 out of 40) collected on flowers display levels of Ascosphaera contamination, but it is not known if all of these species are Ascosphaera hosts or if some of these species are used only as vectors (Evison et al., 2012). Despite evidence of avoidance of contaminated flowers by bumble bees, the impact of floral pollinator parasites presence on the overall pollinator visitation rate of a plant is not yet well understood. In order to assess the impact of parasite presence on overall pollinator visitation to inflorescences, we sprayed natural inflorescences either with pure water or with a solution of A. apis spores, and we recorded and compared the subsequent visitation of pollinators for each inflorescence.
2.2. Observations Observations occurred from 10 a.m. to 4 p.m. during hot and sunny days between the 29th of June to the 3rd of August 2015 in Greensboro, NC, USA. Three observations sites were chosen based on the presence of a wide variety of flowering plants and pollinator species, two from Greensboro Beautiful (http://www.greensborobeautiful.org/gardens/: Greensboro Arboretum (GA) and Gateway Gardens (GG)) and one from the Greensboro Children's Museum (CM) (http://www.gcmuseum.com/ index.php). The UNCG apiary, where the Ascosphaera apis spores originated, are situated 1.3 km from GA (GA-CM = 5 km; GAGG = 8.5 km), 4.3 km from the CM (CM-GG = 3.9 km) and 7.3 km from GG. Flowering plant species were chosen when inflorescence number was sufficient to apply both the treatment and control and after seeing at least one pollinator feed on it. Four plant species were chosen in GA and GG, and one in CM (GA: Echinacea purpurea (Asteraceae), Paeonia spp. (Paeoniaceae), Perovskia atriplicifolia (Lamiaceae), and Vitis riparia (Vitaceae); GG: Gaillardia aristata (Asteraceae), Coreopsis lanceolata (Asteraceae), Eryngium yuccifolium (Apiaceae), Salvia nemorosa (Lamiaceae); CM: Anethum graveolens (Apiaceae)). Those plant species are mostly used as ornamental flowers and most of them are native from North America (https://plants.sc.egov.usda.gov/java/; https:// plants.sc.egov.usda.gov/java/factSheet), except for S. nemorosa (central Europe and western Asia), P. atriplicifolia (southwestern and central Asia), and A. graveolens (Eurasia). In each site, twelve inflorescences (six per treatment) were randomly chosen in large patches of the species and received either the spores or the control solution. Consequently, each of the twelve inflorescences were surrounded by non-treated plants of the same species. The choice for each inflorescence to receive either the spores or the control solution was done in order to get the same number of inflorescences with matching overall size and number of opened flowers in both treatment and control. A. apis spores were applied on inflorescences with a spray bottle, applying one spray per inflorescence (∼0.05 ml, e.g. ∼100,000 spores per inflorescence), with the use of a paper sheet around the inflorescence when spraying allowed to avoid the contamination of other inflorescences. To our knowledge, the natural occurrence of A. apis spores on flowers is not known. The same was done on the other inflorescence with only sterile and distilled water as a control. The use of sterile and distilled water as control solution was chosen since uninfected honey bee brood was not available to control for the effect of honey bee brood residuals. Nevertheless, the thorough washing of the spore solution makes it more likely that any effects are likely the results of the spores rather than the residuals of the honey bee brood. Each plant species was observed during 6 h for only one day. Two people recorded pollinator visitation on 3 A. apis spore-sprayed and 3 water-sprayed inflorescences, for a total of 12 inflorescences observed per plant species. Observations started at 10 a.m., 15 min after spraying, to avoid any effects from the disturbance, and ended at 4 p.m. Pollinator visits were recorded for the whole inflorescence, and pollinators visiting multiple flowers within the same inflorescence were therefore counted as one visit. The observer categorized pollinator visits as one of the following: Approaches (pollinator flew around an inflorescence but did not land); Landings (pollinator landed on an inflorescence but was not seen feeding, no proboscis extension or mouth parts reaching pollen); and Feedings (pollinator was observed landing and feeding on an inflorescence). Additionally, the observer identified the genus of the pollinator or took a picture of it for later identification
2. Material and methods 2.1. Preparation of Ascosphaera apis spores solution Ascosphaera apis spores originated from one frame of a honey bee colony from the University of North Carolina at Greensboro (UNCG) apiary, which were stored at – 20 °C, containing different larval stages of chalkbrood mummies. A. apis spores were extracted from chalkbrood mummies (∼50 mummies) based on the in-vivo harvest method following Jensen et al. (2013). The method was modified in order to avoid bacterial and honey bee contamination. After grinding the black mummies, the pellet was cleaned from the rest of the larvae/pupae body and re-suspended and shaken for 5 min in 5 ml of distilled water; then it was again spun and the supernatant was discarded. This step was repeated until no more of the larvae/pupae body could be detected to the naked eye within the pellet. Moreover, we extended the antibiotic exposure time to 30 min to ensure no bacterial contamination. The spore solutions were spun, the supernatant removed, then the spores were re-suspended in 5 ml of distilled water and shaken for 5 min. This step was repeated two times. Finally, the spores were re-suspended in 50
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Fig. 1. Number of feeding events on inflorescences with and without Ascosphaera apis spores for each pollinator genus and each plant species. (A) Number of Feeding Events on inflorescences with and without Ascosphaera apis spores for each pollinator genus in relation to plant species visited. (B) Number of Feeding Events on inflorescences with and without Ascosphaera apis spores for each plant species in relation to pollinator genus visiting it.
the statistical analyses, as approaches (n = 42) and landings (n = 247) were scarcely observed, not allowing an analysis including the pollinator genus and plant species simultaneously. When a pollinator was seen to forage more than once on an inflorescence (n = 190), only its first observation was kept for the analyses to avoid pseudo-replication. Plant species with fewer than 15 records of pollinator visits were removed from analyses (Paeonia spp. (n = 10) and Eryngium yuccifolium (n = 8)). Finally, only the data from pollinator genera with more than 20 recorded visits and observed in more than one plant species were kept. Our analyses included 4 pollinator genera (n = 917): Apis mellifera, Bombus spp., Lasioglossum spp. and Polistes spp. To unravel if the deposition of A. apis spore solution hinders pollinator visitation of an inflorescence, the number of feeding events by
whenever possible. When the observer failed to identify the pollinator genus, the pollinator was categorized by their order or super-family name (unidentified bees (Apoidea: 26), butterflies (Lepidoptera: 29), and flies (Diptera: 36)). In total, 19 pollinator genera have been identified, with most of observations from Apis mellifera, Bombus spp., and Lasioglossum spp. Finally, the observer reported when a pollinator was seen to forage more than once on treated (both contaminated and control) inflorescences. 2.3. Statistical analyses For all statistical analyses the R software was used (R v3.4.0, R Core Team, 2017). Only the data of feeding events (n = 1299) were kept for 51
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each pollinator genus on each flowering plant species was analysed with a Generalized Linear Model (GLM) following a negative binomial distribution. Negative binomial distribution was preferred from Poisson distribution due to over-dispersion of our count data. The GLM included as explanatory variables: the contamination status of the inflorescence, either A. apis spores or control solution sprayed inflorescence, the interaction between contamination status of the inflorescence with the pollinator genus, and the interactions between contamination status of the inflorescence with the plant species. To account for variations in total number of observations from pollinator genera and from plant species, the logarithm of the total number of feeding events per pollinator genus and per plant species were added as offsets in our GLM. Explanatory variables were removed from the full model by backward stepwise deletion using Log-likelihood Ratio Tests (LRTs) (Crawley, 2005). LRT between the best model and the null model, model containing no explanatory variable, is reported. To further understand how the deposition of A. apis spore solution on inflorescences influences pollinator avoidance depending on the pollinator genus, the plant species and their interactions, the value 1 was assigned to feeding events on the water sprayed inflorescence and the value 0 was assigned to feeding events on the A. apis spores sprayed inflorescence. The proportion of non-contaminated inflorescence visitation was analysed with a GLM following a quasi-binomial distribution. The quasi-binomial distribution was used to account for overdispersion. This GLM included as explanatory variables: the pollinator species, flowering plant species, and their interactions. Explanatory variables were removed from the full model by backward stepwise deletion using Log-likelihood Ratio Tests (LRTs) (Crawley, 2005). LRT between the best and the null model, containing no explanatory variable, is reported. Tukey's honest significant difference post-hoc tests were performed to test for significant differences between explanatory variables simultaneously correcting for multiple comparisons, using the multcomp package (Hothorn et al., 2008).
(Table 1). This suggests that the strength of pollinator avoidance depends simultaneously on both the pollinator genus and plant species. 4. Discussion Our study reveals that in a natural environment the recent deposition of Ascosphaera apis spores on flowers negatively affect the subsequent hymenopteran pollinator visitation. Moreover, it seems that the negative impact of A. apis spore solution on inflorescences depends on both the pollinator genus visiting the plant and the plant species where the spores are present (Fig. 2). Indeed, the number of pollinator visits was lower on A. apis spores- than on water-sprayed inflorescences, regardless of pollinator genus and plant species. Nevertheless, the avoidance of contaminated inflorescences significantly differed among pollinator genera and among plant species. While disease avoidance has been shown in foraging bumble bees (Fouks and Lattorff, 2011), our study reveals that parasite presence on inflorescences could lead to reduced overall pollinator visitation to an inflorescence. Such reduced visitation could lead to pollen limitation (Knight et al., 2005; Harder and Aizen, 2010) and consequently decreased reproductive success of the inflorescence (Irwin and Brody, 1998, 1999; Sahli and Conner, 2006, 2007). Indeed, the presence of bacteria in nectar has been shown to result in reduced pollinator visitation leading to decreased pollination success and seed set in Mimulus aurantiacus flowers (Vannette et al., 2013). It is notable that the two plant species the least affected by A. apis presence (E. purpurea and G. aristata) share uniquely common traits (Fig. 2). Both E. purpurea and G. aristata have capitulum (head) inflorescence composed of disk florets topped by long hairs or surrounded by spike-like bracts. Interestingly, parasites can be deposited and picked up at different rates depending on flower shape and architecture (Durrer and Schmid-Hempel, 1998; Graystock et al., 2015). This suggests that some flowers may provide a more effective transmission platform for parasites than others. Transmission could be affected by flower architecture and related increase in physical contact and/or handling time during foraging. The presence of spikes or hair on flowers could reduce the likelihood of parasite deposition and uptake, diminishing the contact surface between plant and pollinators (Fig. 3). When feeding on E. purpurea, bumble bees, honey bees, and butterflies contacted only the spikes' tips of the capitulum inflorescence with their tarsi (Fig. 3). Such reduced contact with floral parts, leading to a low parasite transmission rate, could result in a low risk of infection and therefore avoidance of contaminated inflorescence may not be as beneficial for a pollinator. Nevertheless, our study suffers some caveats, which does not allow to draw definitive conclusions. Indeed, the number of A. apis spores (∼100,000 spores) deposited on the inflorescence could be high and may not reflect natural levels of A. apis spores on flowers. To our knowledge, the actual levels of A. apis spores on flowers is not known, however many pollinators display levels of Ascosphaera contamination (Evison et al., 2012) and spread of parasites through shared-use of flowers between pollinators is high (Graystock et al., 2015). Moreover, our results could be due to the presence of honey bee brood residuals, since we could not use a pure solution of A. apis spores. Bumble bees are known to avoid flowers with the scent of dead bumble bees, and therefore residues of dead honey bee brood could lead pollinators to avoid flowers (Abbott, 2006). Nonetheless, our A. apis spores solution was thoroughly washed, which likely resulted in undetectable (and very low) levels of honey bee brood residuals. While our study is not sufficient to provide decisive evidence on the impact of A. apis spores on overall visitation of flowering plants, it has the merit to raise the importance of studying the impact of pollinator parasites on plant fitness. Like most animals (Hart, 2011; de Roode and Lefèvre, 2012; Curtis, 2014), pollinators have adapted their foraging behavior to avoid parasites present on flowers (Fouks and Lattorff, 2011). The impact of parasites on ecological interactions, through trait-
3. Results Overall, the deposition of A. apis spores on inflorescences reduced the number of feeding events by pollinators. Indeed, the best GLM model included only the factor inflorescence contamination (LRT: df = 1, χ2 = 4.15, P < 0.05). Despite variations in number of feeding events between water and A. apis spores sprayed inflorescences among pollinator genera and plant species (Fig. 1), the number of feeding events did not significantly differ among pollinator genera and plant species after correcting for total number of feeding events. This may be due to our unbalanced design and low sample size to account for differences among pollinator genera and plant species. Indeed, a power analysis, using lmSupport R package (Curtin, 2018), revealed that our sample size is sufficient to account for differences among pollinator genera (N = 917, df = 6, power = 0.926), but not among plant species (N = 917, df = 12, power = 0.198). The avoidance of contaminated inflorescences was simultaneously dependent on both pollinator genus and plant species (Fig. 2). The best GLM model included the pollinator genus, plant species and their interactions (LRT: df = 17, χ2 = 139.34, P < 0.001). All the factors within the GLM on the proportion of feeding events on the watersprayed inflorescence were significant (GLM: pollinator genus: df = 3, χ2 = 16.14, P < 0.01, plant species: df = 6, χ2 = 55.29, P < 0.001, interaction between pollinator genus and plant species: df = 8, χ2 = 26.55, P < 0.001). Honey bees were more prone to avoid the A. apis sprayed on Vitis riparia inflorescences compared to bumble bees (Table 1). Honey bees and bumble bees avoided more strongly A. apis sprayed on Salvia nemorosa inflorescences compare to solitary bees (Table 1). In addition, bumble bees avoided more strongly A. apis spores when sprayed on Salvia nemorosa than on other plant species (Table 1). Bumble bees also performed better at avoiding A. apis spores when sprayed on Vitis riparia compared to Gaillardia aristata inflorescences 52
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Fig. 2. Proportion of feedings events on the non-contaminated inflorescence (Pollinator Avoidance Efficiency) in relation to flowering plant species and pollinator genus. The bars represent the means of avoidance efficiency for each pollinator genus on each plant species and their 95% confidence interval, the thick black line represent the overall mean avoidance efficiency for each plant species, the numbers at the bottom of each column represents the total feeding events recorded, and the dashed line represents the threshold of avoidance behavior.
Table 1 Results of the pairwise comparisons using post-hoc Tukey's Tests from the Generalized Linear Model on the proportion of non-contaminated inflorescence visitation. Comparisons are made between pollinator genera and plant species (double entry table) within plant species and pollinator genus (name provided before the results), respectively. Significant results are highlighted in bold type. Pollinators
Bombus
Lasioglossum
Polistes
Apis
Salvia: z = 0.605, P = 1 Vitis: z = 2.654, P < 0.05
Anethum: z = 0.359, P = 1 Coreopsis: z = 0.01, P = 1 Salvia: z = 2.826, P < 0.05 Echinacea: z = 0.021, P = 1 Gaillardia: z = −2.174, P = 0.14 Salvia: z = 2.749, P < 0.05
Anethum: z = −0.021, P = 1 Salvia: z = 0.017, P = 1 Vitis: z = 0.683, P = 1 Salvia: z = −0.018, P = 1 Vitis: z = 0.211, P = 1
Bombus
Anethum: z = −0.021, P = 1 Salvia: z = −0.021, P = 1
Lasioglossum
Plants
Coreopsis
Echinacea
Gaillardia
Perovskia
Salvia
Vitis
Anethum
Apis: z = 0.009, P = 1 Lasioglossum: z = 1.221, P = 0.59
Lasioglossum: z = −0.019, P = 1
Lasioglossum: z = 1.743, P = 0.31
Apis: z = 1.711, P = 0.32
Apis: z = 2.035, P = 0.19 Polistes: z = 0.024, P = 1
Lasioglossum: z = 0.02, P=1
Lasioglossum: z = 1.598, P = 0.36
Apis: z = 0.009, P=1
Apis: z = 0.45, P = 1 Lasioglossum: z = 1.998, P = 0.19 Polistes: z = 0, P = 1 Apis: z = 0.009, P = 1 Lasioglossum: z = 1.544, P = 0.38 Bombus: z = -4.75, P < 0.001 Lasioglossum: z = 0.022, P=1 Bombus: z = -6.154, P < 0.001 Lasioglossum: z = 0.956, P = 0.8 Apis: z = −1.333, P = 0.51
Coreopsis
Echinacea
Bombus: z = 2.28, P = 0.11 Lasioglossum: z = 0.021, P=1
Gaillardia
Perovskia Salvia
Apis: z = 0.009, P = 1
Bombus: z = −1.557, P = 0.38
Bombus: z = -3.457, P < 0.01
Apis: z = −0.095, P = 1 Apis: z = 1.631, P = 0.36 Bombus: z = 3.434, P < 0.01 Polistes:z = 0.02, P = 1
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Fig. 3. Photographs of 4 pollinator species visiting Echinacea purpurea inflorescence. Pollinators make contact with the plant mostly through their tarsi and the spikes' tips of the inflorescence. Top-left: Lasioglossum spp. feeding on Echinacea purpurea inflorescence. Top-right: Atalopedes spp. feeding on Echinacea purpurea inflorescence. Bottom-left: Bombus spp. feeding on Echinacea purpurea inflorescence. Bottom-right: Apis mellifera feeding on Echinacea purpurea inflorescence.
mediated interactions, has been documented and is thought to significantly shape ecological communities (Hatcher et al., 2012, 2014; Biere and Bennett, 2013). Therefore, disease avoidance exhibited by foraging pollinators could bear significant impact on plant-pollinator interactions. Further investigations are needed to understand how pollinator parasites may impact plant fitness and plant-pollinator interactions in both wild plants and entomophilous crops.
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Acknowledgments We would like to acknowledge the staff at the Greensboro Arboretum, the Children's Museum and the Gateway Gardens for allowing us to conduct this experiment. We also would like to thank O. Rueppell for providing us with chalkbrood. We would like to thank E. Sakaltaş Ariyak for her help in identifying pollinator species. We would like to thank 3 anonymous reviewers for their helpful comments improving the manuscript quality. We finally would like to thank all members of the Social Insect Lab at the University of North Carolina at Greensboro for their guidance and support. B.F. would like to thank the Union Populaire Républicaine (www.upr.fr) for their moral support. All the facilities and resources used during this experiment were graciously offered by Dr. Rueppell from the University of North Carolina at Greensboro and the staff from Greensboro Arboretum, Children's Museum and Gateway Gardens. References Abbott, K.R., 2006. Bumblebees avoid flowers containing evidence of past predation events. Can. J. Zool. 84, 1240–1247. http://doi.org/10.1139/z06-117. Bailes, E.J., Deutsch, K.R., Bagi, J., Rondissone, L., Brown, M.J.F., Owen, T., 2018. First detection of honey bee viruses in hoverfly (syrphid) pollinators. Biol. Lett. 14, 20180001. https://doi.org/10.1098/rsbl.2018.0001.
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The presence of a larval honey bee parasite, Ascosphaera apis, on flowers reduces pollinator visitation to several plant species
T
Babak Yousefi, Bertrand Fouks∗ Department of Biology, University of North Carolina at Greensboro, 321 McIver Street, Greensboro, NC, 27412, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Plant-pollinator interactions Behavioral immunity Avoidance behavior Host-parasite interactions Tripartite interactions Floral evolution
Many floral traits have evolved through pollinator-mediated selection. In selecting flowers for forage, pollinators rely on multi-modal flower signals and innate preferences. However, pollinators are known for their flexible foraging behavior which varies with experience and numerous cues, such as the presence of competitors and floral resource availability. The shared use of flowers by many pollinators leads to high rate of parasite transmission among pollinators, and flowers can be seen as hot-spots of parasite dispersal. Throughout the animal kingdom, behavioral adaptations enable avoidance of parasite exposure. Bumblebees are known to avoid contaminated flowers, however nothing is known regarding the impact of parasite presence on overall pollinator visitation. In this study, we measured the effect of a larval honey bee parasite, the fungus Ascosphaera apis causing chalkbrood disease, on hymenopteran pollinator visitation of several plant species in a natural setting. Several contaminated flowering plant species suffered not only the subsequent reduction of visitation by honey bees but also the subsequent reduction of their overall pollinator visitation. Two plant species with similar flower architecture were the least impacted by the presence of the spores. This study highlights the negative impact of the presence of pollinator parasites for plants and its possible consequences for floral evolution.
1. Introduction Angiosperms have evolved interactions with animals to facilitate their reproduction (Dilcher, 2000). Pollinator-mediated selection resulting from those interactions is a mechanism underlying floral diversity (Fenster et al., 2004; Sapir and Armbruster, 2011). In theory, pollinator-mediated selection favors floral traits that maximize reproductive fitness via pollen export and import (Morgan, 1992). Thus many floral traits have evolved to attract pollinators (Fenster et al., 2004; Schiestl and Johnson, 2013) and pollinators rely on innate preferences (Goulson et al., 2007) and associative learning to forage on flowers (Chittka and Thomson, 2001). Moreover, pollinators rely on multi-modal signals, such floral color and scent, to choose flowers (Leonard et al., 2011a; Leonard and Masek, 2014). Pollinators also exhibit flexibility in their foraging behavior as they gain experience (Riffell et al., 2014). This leads to complex decision making and behaviors of pollinators relying on multi-modal signals to choose flowers to forage (Chittka and Thomson, 2001; Leonard et al., 2011b), in order to optimize their exploitation of floral resources. Parasites are of major ecological and evolutionary importance (Hatcher and Dunn, 2011), and have been observed influencing diet
choices in many host species (Hart, 2011; de Roode and Lefèvre, 2012; Curtis, 2014). Parasites and related diseases represent a significant threat, reducing organismal reproduction and lifespan (Bonsall, 2004), and acting as a strong evolutionary force (Bell and Maynard Smith, 1987). Moreover, parasites can indirectly impact several aspects of ecological relationships (Wood and Johnson, 2015), including trophic (Lafferty et al., 2006, 2008), competitive (Hatcher et al., 2006) and mutualistic interactions (Liere and Larsen, 2010; Wood et al., 2018). Parasite threat is inherently dependent on the likelihood of parasite encounter and transmission. Shared use of resources plays a major role in the dynamics of parasite dispersal (Prüss-Ustün et al., 2002). Since parasites often populate environments in which they encounter numerous species, non-host species can also be important in parasite dispersal (Rigaud et al., 2010; Dunn and Perkins, 2012). Parasites transmitted among species can be dispersed widely and to novel locations (Tatem et al., 2006). Flowers have been shown to play a role in parasite transmission among pollinator species, with flowers acting as hot-spots of disease dispersal (Durrer and Schmid-Hempel, 1998; Graystock et al., 2015). While some plant-pollinator systems are specialized, the vast majority of flowering plant species are visited by multiple pollinator species in a complex web of interactions (Waser
∗ Corresponding author. Present address: Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, P. O. Box 1066 Blindern, NO-0316, Oslo, Norway. E-mail address: [email protected] (B. Fouks).
https://doi.org/10.1016/j.actao.2019.03.006 Received 6 December 2018; Received in revised form 26 February 2019; Accepted 18 March 2019 1146-609X/ © 2019 Elsevier Masson SAS. All rights reserved.
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1 ml of distilled water and the number of spores was counted at a 10−3 dilution in a phase hematocytometer (Hausser Scientific®, Horsham, PA, USA). Spore solutions were combined to obtain a concentration of 2 × 106 spores/ml (for a total of 20 ml). Spore solutions were either made immediately before observations or were stored at 6 °C in the dark until use (max. 4 days).
et al., 1996; Fontaine et al., 2006). The vectoring of parasites by nonhost pollinator species during shared flower use may be of great importance in pollinator–parasite interactions (Evison et al., 2012; McArt et al., 2014), and there is increasing evidence for the incidence and impact of parasite transmission among pollinator taxa (Evison et al., 2012; Fürst et al., 2014; McMahon et al., 2015; Bailes et al., 2018). The selective pressure parasites put on their hosts drives the adaptation of a wide range of defense mechanisms (Schmid-Hempel, 2011). One of these mechanisms is the adaptation of host behavior, with behavioral avoidance (e.g. physical avoidance) being the most important and wide-ranging strategy in the animal kingdom (Hart, 2011). Behavioral avoidance is the first line of defense against infection, as it can prevent or reduce parasite encounter (Schmid-Hempel, 2011), and is thus likely to be a highly cost-effective form of defense. Few studies demonstrate the impact of parasites on foraging pollinator decisionmaking and flower choice (Fouks and Lattorff, 2011; Richardson et al., 2016). Behavioral avoidance of flowers contaminated by parasites was first observed in bumble bees (Fouks and Lattorff, 2011). Bumble bees reduced their number of visits and feeding duration on contaminated flowers (Fouks and Lattorff, 2011, 2013). Avoidance of parasite-infested flowers may negatively impact plant fitness; as reduced pollinator visitation can lead to pollen limitation (Knight et al., 2005; Harder and Aizen, 2010) and decreased plant reproductive success (Irwin and Brody, 1998, 1999; Sahli and Conner, 2006, 2007). The fungus Ascosphaera apis is a common parasite of honey bees, provoking chalkbrood disease and killing larvae (Evison and Jensen, 2018). Evidence suggests A. apis may be transmitted horizontally through large number of pollinator species via the shared use of flowers (Evison et al., 2012; Mc Art et al., 2014). While A. apis is specific to honey bee brood, it is also known of cross-species transmission to the brood of some wild bees (i.e. Xylocopa californica and Megachile rotundata), and the adult stage of bumble bees (Maxfield-Taylor et al., 2015; Evison and Jensen, 2018). Furthermore, honey bees (2 individuals out of 12), bumble bees (34 out of 114), solitary bees (50 out of 56), wasps (27 out of 53) and hover-flies (26 out of 40) collected on flowers display levels of Ascosphaera contamination, but it is not known if all of these species are Ascosphaera hosts or if some of these species are used only as vectors (Evison et al., 2012). Despite evidence of avoidance of contaminated flowers by bumble bees, the impact of floral pollinator parasites presence on the overall pollinator visitation rate of a plant is not yet well understood. In order to assess the impact of parasite presence on overall pollinator visitation to inflorescences, we sprayed natural inflorescences either with pure water or with a solution of A. apis spores, and we recorded and compared the subsequent visitation of pollinators for each inflorescence.
2.2. Observations Observations occurred from 10 a.m. to 4 p.m. during hot and sunny days between the 29th of June to the 3rd of August 2015 in Greensboro, NC, USA. Three observations sites were chosen based on the presence of a wide variety of flowering plants and pollinator species, two from Greensboro Beautiful (http://www.greensborobeautiful.org/gardens/: Greensboro Arboretum (GA) and Gateway Gardens (GG)) and one from the Greensboro Children's Museum (CM) (http://www.gcmuseum.com/ index.php). The UNCG apiary, where the Ascosphaera apis spores originated, are situated 1.3 km from GA (GA-CM = 5 km; GAGG = 8.5 km), 4.3 km from the CM (CM-GG = 3.9 km) and 7.3 km from GG. Flowering plant species were chosen when inflorescence number was sufficient to apply both the treatment and control and after seeing at least one pollinator feed on it. Four plant species were chosen in GA and GG, and one in CM (GA: Echinacea purpurea (Asteraceae), Paeonia spp. (Paeoniaceae), Perovskia atriplicifolia (Lamiaceae), and Vitis riparia (Vitaceae); GG: Gaillardia aristata (Asteraceae), Coreopsis lanceolata (Asteraceae), Eryngium yuccifolium (Apiaceae), Salvia nemorosa (Lamiaceae); CM: Anethum graveolens (Apiaceae)). Those plant species are mostly used as ornamental flowers and most of them are native from North America (https://plants.sc.egov.usda.gov/java/; https:// plants.sc.egov.usda.gov/java/factSheet), except for S. nemorosa (central Europe and western Asia), P. atriplicifolia (southwestern and central Asia), and A. graveolens (Eurasia). In each site, twelve inflorescences (six per treatment) were randomly chosen in large patches of the species and received either the spores or the control solution. Consequently, each of the twelve inflorescences were surrounded by non-treated plants of the same species. The choice for each inflorescence to receive either the spores or the control solution was done in order to get the same number of inflorescences with matching overall size and number of opened flowers in both treatment and control. A. apis spores were applied on inflorescences with a spray bottle, applying one spray per inflorescence (∼0.05 ml, e.g. ∼100,000 spores per inflorescence), with the use of a paper sheet around the inflorescence when spraying allowed to avoid the contamination of other inflorescences. To our knowledge, the natural occurrence of A. apis spores on flowers is not known. The same was done on the other inflorescence with only sterile and distilled water as a control. The use of sterile and distilled water as control solution was chosen since uninfected honey bee brood was not available to control for the effect of honey bee brood residuals. Nevertheless, the thorough washing of the spore solution makes it more likely that any effects are likely the results of the spores rather than the residuals of the honey bee brood. Each plant species was observed during 6 h for only one day. Two people recorded pollinator visitation on 3 A. apis spore-sprayed and 3 water-sprayed inflorescences, for a total of 12 inflorescences observed per plant species. Observations started at 10 a.m., 15 min after spraying, to avoid any effects from the disturbance, and ended at 4 p.m. Pollinator visits were recorded for the whole inflorescence, and pollinators visiting multiple flowers within the same inflorescence were therefore counted as one visit. The observer categorized pollinator visits as one of the following: Approaches (pollinator flew around an inflorescence but did not land); Landings (pollinator landed on an inflorescence but was not seen feeding, no proboscis extension or mouth parts reaching pollen); and Feedings (pollinator was observed landing and feeding on an inflorescence). Additionally, the observer identified the genus of the pollinator or took a picture of it for later identification
2. Material and methods 2.1. Preparation of Ascosphaera apis spores solution Ascosphaera apis spores originated from one frame of a honey bee colony from the University of North Carolina at Greensboro (UNCG) apiary, which were stored at – 20 °C, containing different larval stages of chalkbrood mummies. A. apis spores were extracted from chalkbrood mummies (∼50 mummies) based on the in-vivo harvest method following Jensen et al. (2013). The method was modified in order to avoid bacterial and honey bee contamination. After grinding the black mummies, the pellet was cleaned from the rest of the larvae/pupae body and re-suspended and shaken for 5 min in 5 ml of distilled water; then it was again spun and the supernatant was discarded. This step was repeated until no more of the larvae/pupae body could be detected to the naked eye within the pellet. Moreover, we extended the antibiotic exposure time to 30 min to ensure no bacterial contamination. The spore solutions were spun, the supernatant removed, then the spores were re-suspended in 5 ml of distilled water and shaken for 5 min. This step was repeated two times. Finally, the spores were re-suspended in 50
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Fig. 1. Number of feeding events on inflorescences with and without Ascosphaera apis spores for each pollinator genus and each plant species. (A) Number of Feeding Events on inflorescences with and without Ascosphaera apis spores for each pollinator genus in relation to plant species visited. (B) Number of Feeding Events on inflorescences with and without Ascosphaera apis spores for each plant species in relation to pollinator genus visiting it.
the statistical analyses, as approaches (n = 42) and landings (n = 247) were scarcely observed, not allowing an analysis including the pollinator genus and plant species simultaneously. When a pollinator was seen to forage more than once on an inflorescence (n = 190), only its first observation was kept for the analyses to avoid pseudo-replication. Plant species with fewer than 15 records of pollinator visits were removed from analyses (Paeonia spp. (n = 10) and Eryngium yuccifolium (n = 8)). Finally, only the data from pollinator genera with more than 20 recorded visits and observed in more than one plant species were kept. Our analyses included 4 pollinator genera (n = 917): Apis mellifera, Bombus spp., Lasioglossum spp. and Polistes spp. To unravel if the deposition of A. apis spore solution hinders pollinator visitation of an inflorescence, the number of feeding events by
whenever possible. When the observer failed to identify the pollinator genus, the pollinator was categorized by their order or super-family name (unidentified bees (Apoidea: 26), butterflies (Lepidoptera: 29), and flies (Diptera: 36)). In total, 19 pollinator genera have been identified, with most of observations from Apis mellifera, Bombus spp., and Lasioglossum spp. Finally, the observer reported when a pollinator was seen to forage more than once on treated (both contaminated and control) inflorescences. 2.3. Statistical analyses For all statistical analyses the R software was used (R v3.4.0, R Core Team, 2017). Only the data of feeding events (n = 1299) were kept for 51
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each pollinator genus on each flowering plant species was analysed with a Generalized Linear Model (GLM) following a negative binomial distribution. Negative binomial distribution was preferred from Poisson distribution due to over-dispersion of our count data. The GLM included as explanatory variables: the contamination status of the inflorescence, either A. apis spores or control solution sprayed inflorescence, the interaction between contamination status of the inflorescence with the pollinator genus, and the interactions between contamination status of the inflorescence with the plant species. To account for variations in total number of observations from pollinator genera and from plant species, the logarithm of the total number of feeding events per pollinator genus and per plant species were added as offsets in our GLM. Explanatory variables were removed from the full model by backward stepwise deletion using Log-likelihood Ratio Tests (LRTs) (Crawley, 2005). LRT between the best model and the null model, model containing no explanatory variable, is reported. To further understand how the deposition of A. apis spore solution on inflorescences influences pollinator avoidance depending on the pollinator genus, the plant species and their interactions, the value 1 was assigned to feeding events on the water sprayed inflorescence and the value 0 was assigned to feeding events on the A. apis spores sprayed inflorescence. The proportion of non-contaminated inflorescence visitation was analysed with a GLM following a quasi-binomial distribution. The quasi-binomial distribution was used to account for overdispersion. This GLM included as explanatory variables: the pollinator species, flowering plant species, and their interactions. Explanatory variables were removed from the full model by backward stepwise deletion using Log-likelihood Ratio Tests (LRTs) (Crawley, 2005). LRT between the best and the null model, containing no explanatory variable, is reported. Tukey's honest significant difference post-hoc tests were performed to test for significant differences between explanatory variables simultaneously correcting for multiple comparisons, using the multcomp package (Hothorn et al., 2008).
(Table 1). This suggests that the strength of pollinator avoidance depends simultaneously on both the pollinator genus and plant species. 4. Discussion Our study reveals that in a natural environment the recent deposition of Ascosphaera apis spores on flowers negatively affect the subsequent hymenopteran pollinator visitation. Moreover, it seems that the negative impact of A. apis spore solution on inflorescences depends on both the pollinator genus visiting the plant and the plant species where the spores are present (Fig. 2). Indeed, the number of pollinator visits was lower on A. apis spores- than on water-sprayed inflorescences, regardless of pollinator genus and plant species. Nevertheless, the avoidance of contaminated inflorescences significantly differed among pollinator genera and among plant species. While disease avoidance has been shown in foraging bumble bees (Fouks and Lattorff, 2011), our study reveals that parasite presence on inflorescences could lead to reduced overall pollinator visitation to an inflorescence. Such reduced visitation could lead to pollen limitation (Knight et al., 2005; Harder and Aizen, 2010) and consequently decreased reproductive success of the inflorescence (Irwin and Brody, 1998, 1999; Sahli and Conner, 2006, 2007). Indeed, the presence of bacteria in nectar has been shown to result in reduced pollinator visitation leading to decreased pollination success and seed set in Mimulus aurantiacus flowers (Vannette et al., 2013). It is notable that the two plant species the least affected by A. apis presence (E. purpurea and G. aristata) share uniquely common traits (Fig. 2). Both E. purpurea and G. aristata have capitulum (head) inflorescence composed of disk florets topped by long hairs or surrounded by spike-like bracts. Interestingly, parasites can be deposited and picked up at different rates depending on flower shape and architecture (Durrer and Schmid-Hempel, 1998; Graystock et al., 2015). This suggests that some flowers may provide a more effective transmission platform for parasites than others. Transmission could be affected by flower architecture and related increase in physical contact and/or handling time during foraging. The presence of spikes or hair on flowers could reduce the likelihood of parasite deposition and uptake, diminishing the contact surface between plant and pollinators (Fig. 3). When feeding on E. purpurea, bumble bees, honey bees, and butterflies contacted only the spikes' tips of the capitulum inflorescence with their tarsi (Fig. 3). Such reduced contact with floral parts, leading to a low parasite transmission rate, could result in a low risk of infection and therefore avoidance of contaminated inflorescence may not be as beneficial for a pollinator. Nevertheless, our study suffers some caveats, which does not allow to draw definitive conclusions. Indeed, the number of A. apis spores (∼100,000 spores) deposited on the inflorescence could be high and may not reflect natural levels of A. apis spores on flowers. To our knowledge, the actual levels of A. apis spores on flowers is not known, however many pollinators display levels of Ascosphaera contamination (Evison et al., 2012) and spread of parasites through shared-use of flowers between pollinators is high (Graystock et al., 2015). Moreover, our results could be due to the presence of honey bee brood residuals, since we could not use a pure solution of A. apis spores. Bumble bees are known to avoid flowers with the scent of dead bumble bees, and therefore residues of dead honey bee brood could lead pollinators to avoid flowers (Abbott, 2006). Nonetheless, our A. apis spores solution was thoroughly washed, which likely resulted in undetectable (and very low) levels of honey bee brood residuals. While our study is not sufficient to provide decisive evidence on the impact of A. apis spores on overall visitation of flowering plants, it has the merit to raise the importance of studying the impact of pollinator parasites on plant fitness. Like most animals (Hart, 2011; de Roode and Lefèvre, 2012; Curtis, 2014), pollinators have adapted their foraging behavior to avoid parasites present on flowers (Fouks and Lattorff, 2011). The impact of parasites on ecological interactions, through trait-
3. Results Overall, the deposition of A. apis spores on inflorescences reduced the number of feeding events by pollinators. Indeed, the best GLM model included only the factor inflorescence contamination (LRT: df = 1, χ2 = 4.15, P < 0.05). Despite variations in number of feeding events between water and A. apis spores sprayed inflorescences among pollinator genera and plant species (Fig. 1), the number of feeding events did not significantly differ among pollinator genera and plant species after correcting for total number of feeding events. This may be due to our unbalanced design and low sample size to account for differences among pollinator genera and plant species. Indeed, a power analysis, using lmSupport R package (Curtin, 2018), revealed that our sample size is sufficient to account for differences among pollinator genera (N = 917, df = 6, power = 0.926), but not among plant species (N = 917, df = 12, power = 0.198). The avoidance of contaminated inflorescences was simultaneously dependent on both pollinator genus and plant species (Fig. 2). The best GLM model included the pollinator genus, plant species and their interactions (LRT: df = 17, χ2 = 139.34, P < 0.001). All the factors within the GLM on the proportion of feeding events on the watersprayed inflorescence were significant (GLM: pollinator genus: df = 3, χ2 = 16.14, P < 0.01, plant species: df = 6, χ2 = 55.29, P < 0.001, interaction between pollinator genus and plant species: df = 8, χ2 = 26.55, P < 0.001). Honey bees were more prone to avoid the A. apis sprayed on Vitis riparia inflorescences compared to bumble bees (Table 1). Honey bees and bumble bees avoided more strongly A. apis sprayed on Salvia nemorosa inflorescences compare to solitary bees (Table 1). In addition, bumble bees avoided more strongly A. apis spores when sprayed on Salvia nemorosa than on other plant species (Table 1). Bumble bees also performed better at avoiding A. apis spores when sprayed on Vitis riparia compared to Gaillardia aristata inflorescences 52
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Fig. 2. Proportion of feedings events on the non-contaminated inflorescence (Pollinator Avoidance Efficiency) in relation to flowering plant species and pollinator genus. The bars represent the means of avoidance efficiency for each pollinator genus on each plant species and their 95% confidence interval, the thick black line represent the overall mean avoidance efficiency for each plant species, the numbers at the bottom of each column represents the total feeding events recorded, and the dashed line represents the threshold of avoidance behavior.
Table 1 Results of the pairwise comparisons using post-hoc Tukey's Tests from the Generalized Linear Model on the proportion of non-contaminated inflorescence visitation. Comparisons are made between pollinator genera and plant species (double entry table) within plant species and pollinator genus (name provided before the results), respectively. Significant results are highlighted in bold type. Pollinators
Bombus
Lasioglossum
Polistes
Apis
Salvia: z = 0.605, P = 1 Vitis: z = 2.654, P < 0.05
Anethum: z = 0.359, P = 1 Coreopsis: z = 0.01, P = 1 Salvia: z = 2.826, P < 0.05 Echinacea: z = 0.021, P = 1 Gaillardia: z = −2.174, P = 0.14 Salvia: z = 2.749, P < 0.05
Anethum: z = −0.021, P = 1 Salvia: z = 0.017, P = 1 Vitis: z = 0.683, P = 1 Salvia: z = −0.018, P = 1 Vitis: z = 0.211, P = 1
Bombus
Anethum: z = −0.021, P = 1 Salvia: z = −0.021, P = 1
Lasioglossum
Plants
Coreopsis
Echinacea
Gaillardia
Perovskia
Salvia
Vitis
Anethum
Apis: z = 0.009, P = 1 Lasioglossum: z = 1.221, P = 0.59
Lasioglossum: z = −0.019, P = 1
Lasioglossum: z = 1.743, P = 0.31
Apis: z = 1.711, P = 0.32
Apis: z = 2.035, P = 0.19 Polistes: z = 0.024, P = 1
Lasioglossum: z = 0.02, P=1
Lasioglossum: z = 1.598, P = 0.36
Apis: z = 0.009, P=1
Apis: z = 0.45, P = 1 Lasioglossum: z = 1.998, P = 0.19 Polistes: z = 0, P = 1 Apis: z = 0.009, P = 1 Lasioglossum: z = 1.544, P = 0.38 Bombus: z = -4.75, P < 0.001 Lasioglossum: z = 0.022, P=1 Bombus: z = -6.154, P < 0.001 Lasioglossum: z = 0.956, P = 0.8 Apis: z = −1.333, P = 0.51
Coreopsis
Echinacea
Bombus: z = 2.28, P = 0.11 Lasioglossum: z = 0.021, P=1
Gaillardia
Perovskia Salvia
Apis: z = 0.009, P = 1
Bombus: z = −1.557, P = 0.38
Bombus: z = -3.457, P < 0.01
Apis: z = −0.095, P = 1 Apis: z = 1.631, P = 0.36 Bombus: z = 3.434, P < 0.01 Polistes:z = 0.02, P = 1
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B. Yousefi and B. Fouks
Fig. 3. Photographs of 4 pollinator species visiting Echinacea purpurea inflorescence. Pollinators make contact with the plant mostly through their tarsi and the spikes' tips of the inflorescence. Top-left: Lasioglossum spp. feeding on Echinacea purpurea inflorescence. Top-right: Atalopedes spp. feeding on Echinacea purpurea inflorescence. Bottom-left: Bombus spp. feeding on Echinacea purpurea inflorescence. Bottom-right: Apis mellifera feeding on Echinacea purpurea inflorescence.
mediated interactions, has been documented and is thought to significantly shape ecological communities (Hatcher et al., 2012, 2014; Biere and Bennett, 2013). Therefore, disease avoidance exhibited by foraging pollinators could bear significant impact on plant-pollinator interactions. Further investigations are needed to understand how pollinator parasites may impact plant fitness and plant-pollinator interactions in both wild plants and entomophilous crops.
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Acknowledgments We would like to acknowledge the staff at the Greensboro Arboretum, the Children's Museum and the Gateway Gardens for allowing us to conduct this experiment. We also would like to thank O. Rueppell for providing us with chalkbrood. We would like to thank E. Sakaltaş Ariyak for her help in identifying pollinator species. We would like to thank 3 anonymous reviewers for their helpful comments improving the manuscript quality. We finally would like to thank all members of the Social Insect Lab at the University of North Carolina at Greensboro for their guidance and support. B.F. would like to thank the Union Populaire Républicaine (www.upr.fr) for their moral support. All the facilities and resources used during this experiment were graciously offered by Dr. Rueppell from the University of North Carolina at Greensboro and the staff from Greensboro Arboretum, Children's Museum and Gateway Gardens. References Abbott, K.R., 2006. Bumblebees avoid flowers containing evidence of past predation events. Can. J. Zool. 84, 1240–1247. http://doi.org/10.1139/z06-117. Bailes, E.J., Deutsch, K.R., Bagi, J., Rondissone, L., Brown, M.J.F., Owen, T., 2018. First detection of honey bee viruses in hoverfly (syrphid) pollinators. Biol. Lett. 14, 20180001. https://doi.org/10.1098/rsbl.2018.0001.
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