Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization.

Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and the fitness of overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.

Pollen Diet Diversity does not Affect Gut Bacterial Communities or Melanization in a Social and Solitary Bee Species.

Pollinators face many stressors, including reduced floral diversity. A low-diversity diet can impair organisms' ability to cope with additional stressors, such as pathogens, by altering the gut microbiome and/or immune function, but these effects are understudied for most pollinators. We investigated the impact of pollen diet diversity on two ecologically and economically important generalist pollinators, the social bumble bee (Bombus impatiens) and the solitary alfalfa leafcutter bee (Megachile rotundata). We experimentally tested the effect of one-, two-, or three-species pollen diets on gut bacterial communities in both species, and the melanization immune response in B. impatiens. Pollen diets included dandelion (Taraxacum officinale), staghorn sumac (Rhus typhina), and hawthorn (Crataegus sp.) alone, each pair-wise combination, or a mix of all three species. We fed bees their diet for 7 days and then dissected out guts and sequenced 16S rRNA gene amplicons to characterize gut bacterial communities. To assess melanization in B. impatiens, we inserted microfilament implants into the bee abdomen and measured melanin deposition on the implant. We found that pollen diet did not influence gut bacterial communities in M. rotundata. In B. impatiens, pollen diet composition, but not diversity, affected gut bacterial richness in older, but not newly-emerged bees. Pollen diet did not affect the melanization response in B. impatiens. Our results suggest that even a monofloral, low-quality pollen diet such as dandelion can support diverse gut bacterial communities in captive-reared adults of these bee species. These findings shed light on the effects of reduced diet diversity on bee health.

Gut Transplants from Bees Fed an Antipathogenic Pollen Diet Do Not Confer Pathogen Resistance to Recipients.

Pollinators are threatened by diverse stressors, including microbial pathogens such as Crithidia bombi. Consuming sunflower pollen dramatically reduces C. bombi infection in the bumble bee Bombus impatiens, but the mechanism behind this medicinal effect is unclear. We asked whether diet mediates resistance to C. bombi through changes in the gut microbiome. We hypothesized that sunflower pollen changes the gut microbiome, which in turn reduces Crithidia infection. To test this, we performed a gut transplant experiment. We fed donor bees either a sunflower pollen treatment or buckwheat pollen as a control treatment and then inoculated recipient bees with homogenized guts from either sunflower-fed or buckwheat-fed donor bees. All recipient bees were then fed a wildflower pollen diet. Two days after the transplant, we infected recipients with C. bombi, and 2 days later, we provided another donor gut transplant. To quantify infection, we performed both fecal screens and dissections of the recipient bees. We found no significant differences in C. bombi infection intensity or presence between bees that received sunflower-fed microbiomes versus buckwheat-fed microbiomes. This suggests that sunflower pollen's effects on pathogen resistance are not mediated by gut microbiota.

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

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

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

Differential bumble bee gene expression associated with pathogen infection and pollen diet.

BACKGROUND: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. RESULTS: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. CONCLUSIONS: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

The intersection of bee and flower sexes: pollen presence shapes sex-specific bee foraging associations in sunflower.

Foraging preferences are known to differ among bee taxa, and can also differ between male and female bees of the same species. Similarly, bees can prefer a specific flower sex, particularly if only one sex provides pollen. Such variation in foraging preferences could lead to divergent bee communities visiting different flower sexes of a plant species. We sampled bees visiting sunflowers to characterize bee species richness, abundance, and sex ratios on pollen-fertile and pollen-sterile cultivars. We asked whether female or male bees were more abundant on sunflowers, whether female bees were more abundant on pollen-fertile or pollen-sterile cultivars, and whether pollen presence predicted the sex of sampled bees. We further asked whether the bee community differed between pollen-fertile and pollen-sterile cultivars. Females of most bee species were more abundant on sunflowers compared to males, and females were usually more abundant on pollen-fertile cultivars. In three bee species, pollen presence was predictive of a bee's sex, with females more abundant on pollen-fertile cultivars than males. Further, the bee community differed significantly between pollen-fertile and pollen-sterile cultivars, with two bee species functioning as indicators for pollen-fertile sunflowers. Our results demonstrate that a bee's sex shapes foraging associations on sunflowers and influences abundance between pollen-fertile and pollen-sterile cultivars, and that pollen-fertile and pollen-sterile cultivars are visited by different bee communities. Bee sexes and flower pollen presence may be under-appreciated factors shaping pollination services in both agricultural and natural ecosystems, and could be important considerations for pollination of crops with pollen-fertile and pollen-sterile flowers.

Comparative analysis of 3 pollen sterilization methods for feeding bumble bees.

Pollen is an essential component of bee diets, and rearing bumble bees (Bombus spp.) for commercial use necessitates feeding pollen in mass quantities. This pollen is collected from honey bee (Apis mellifera L.) colonies because neither an artificial diet nor an economical, large-scale pollen collection process from flowers is available. The provenance of honey bee-collected pollen is often unknown, and in some cases has crossed international borders. Both deformed wing virus (DWV) and the fungal pathogen Ascosphaera apis (Claussen) Olive & Spiltoir (cause of chalkbrood disease); occur in honey bee-collected pollen, and infections have been observed in bumble bees. We used these pathogens as general surrogates for viruses and spore-forming fungal diseases to test the efficacy of 3 sterilization methods, and assessed whether treatment altered pollen quality for the bumble bee. Using honey bee-collected pollen spiked with known doses of DWV and A. apis, we compared gamma irradiation (GI), ozone fumigation (OZ), and ethylene oxide fumigation (EO) against an untreated positive control and a negative control. Following sterilization treatments, we tested A. apis spore viability, detected viral presence with PCR, and tested palatability to the bumble bee Bombus impatiens Cresson. We also measured bacterial growth from pollens treated with EO and GI. GI and EO outperformed OZ treatment in pathogen suppression. EO had the highest sterilizing properties under commercial conditions and retained palatability and supported bee development better than other treatments. These results suggest that EO sterilization reduces pathogen risks while retaining pollen quality as a food source for rearing bumble bees.

Sunflower-Associated Reductions in Varroa Mite Infestation of Honey Bee Colonies.

Landscapes can affect parasite epidemiology in wild and agricultural animals. Honey bees are threatened by loss of floral resources and by parasites, principally the mite Varroa destructor and the viruses it vectors. Existing mite control relies heavily on chemical treatments that can adversely affect bees. Alternative, pesticide-free control methods are needed to mitigate infestation with these ectoparasites. Many flowering plants provide nectar and pollen that confer resistance to parasites. Enrichment of landscapes with antiparasitic floral resources could therefore provide a sustainable means of parasite control in pollinators. Floral rewards of Asteraceae plants can reduce parasitic infection in diverse bee species, including honey and bumble bees. Here, we tested the effects of sunflower (Helianthus annuus) cropland and pollen supplementation on honey bee resistance to macro- and microparasites. Although sunflower had nonsignificant effects on microparasites, We found that increased sunflower pollen availability correlated with reduced Varroa mite infestation in landscapes and pollen-supplemented colonies. At the landscape level, each doubling of sunflower crop area was associated with a 28% reduction in mite infestation. In field trials, late-summer supplementation of colonies with sunflower pollen reduced mite infestation by 2.75-fold relative to artificial pollen. United States sunflower crop acreage has declined by 2% per year since 1980, however, suggesting reduced availability of this floral resource. Although further research is needed to determine whether the observed effects represent direct inhibition of mite fecundity or mite-limiting reductions in honey bee brood-rearing, our findings suggest the potential for sunflower plantings or pollen supplements to counteract a major driver of honey bee losses worldwide.

Manipulation of multiple floral traits demonstrates role in pollinator disease transmission.

Plants modulate multitrophic ecological interactions, and variation in plant traits can affect these interactions. Pollinators are exposed to pathogens at flowers and acquire or transmit pathogens at different rates on different plant species, but the traits mediating those interactions are almost entirely unknown. We experimentally manipulated five plant traits that span scales including flower, inflorescence, and plant, to determine their effects on pathogen transmission between foraging bees. Specifically, we manipulated two morphological traits (corolla lip length and flower orientation within an inflorescence) and three resource distribution traits (inflorescence nectar, plant patch nectar, and plant aggregation) in tents to test how plant traits affect bee pathogen transmission. We also quantified foraging behavior and fecal deposition patterns as potential mechanisms driving differences in transmission, and assessed trait manipulation consequences for bee reproduction. We found that pathogen transmission was reduced when we trimmed the corolla lip, evenly dispersed nectar distribution within an inflorescence, or aggregated plants in space. Some traits also affected bee reproduction; tents with trimmed corollas had more larval production than control tents, and tents with evenly distributed nectar across plant patches had more larval production than tents with clumped resources. Thus, some trait manipulations both reduced transmission and increased bee microcolony reproduction, although our design does not allow us to discern whether these are related or separate effects. Taken together, our results demonstrate causal effects of several floral traits on pathogen transmission and pollinator reproduction, indicating the importance of intraspecific plant trait variation for pollinator health and population dynamics.

Pollen limitation of native plant reproduction in an urban landscape.

PREMISE: Evidence suggests that bees may benefit from moderate levels of human development. However, the effects of human development on pollination and reproduction of bee-pollinated plants are less-well understood. Studies have measured natural variation in pollination and plant reproduction as a function of urbanization, but few have experimentally measured the magnitude of pollen limitation in urban vs. non-urban sites. Doing so is important to unambiguously link changes in pollination to plant reproduction. Previous work in the Southeastern United States found that urban sites supported twice the abundance of bees compared to non-urban sites. We tested the hypothesis that greater bee abundance in some of the same urban sites translates into reduced pollen limitation compared to non-urban sites. METHODS: We manipulated pollination to three native, wild-growing, bee-pollinated plants: Gelsemium sempervirens, Oenothera fruticosa, and Campsis radicans. Using supplemental pollinations, we tested for pollen limitation of three components of female reproduction in paired urban and non-urban sites. We also measured pollen receipt as a proxy for pollinator visitation. RESULTS: We found that all three plant species were pollen-limited for some measures of female reproduction. However, opposite to our original hypothesis, two of the three species were more pollen-limited in urban relative to non-urban sites. We found that open-pollinated flowers in urban sites received less conspecific and more heterospecific pollen on average than those in non-urban sites. CONCLUSIONS: These results suggest that even when urban sites have more abundant pollinators, this may not alleviate pollen limitation of native plant reproduction in urban landscapes.

Consuming sunflower pollen reduced pathogen infection but did not alter measures of immunity in bumblebees.

Certain diets can benefit bee health by reducing pathogens, but the mechanism(s) driving these medicinal effects are largely unexplored. Recent research found that sunflower (Helianthus annuus) pollen reduces the gut pathogen Crithidia bombi in the common eastern bumblebee (Bombus impatiens). Here, we tested the effects of sunflower pollen and infection on two bee immune metrics to determine whether sunflower pollen diet drives changes in host immunity that can explain this medicinal effect. Bees were infected with C. bombi or not and given either sunflower or wildflower pollen. Subsequently, bees received a benign immune challenge or were left naive to test the induced and constitutive immune responses, respectively. We measured haemolymph phenoloxidase activity, involved in the melanization cascade, and antibacterial activity. Sunflower pollen reduced C. bombi infection, but we found no significant pollen diet effect on either immune measure. Phenoloxidase activity was also not affected by C. bombi infection status; however, uninfected bees were more likely to have measurable constitutive antibacterial activity, while infected bees had higher induced antibacterial activity. Overall, we found that sunflower pollen does not significantly affect the immune responses we measured, suggesting that the mechanisms underlying its medicinal effect do not involve these bee immune parameters. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Understanding effects of floral products on bee parasites: Mechanisms, synergism, and ecological complexity.

Floral nectar and pollen commonly contain diverse secondary metabolites. While these compounds are classically thought to play a role in plant defense, recent research indicates that they may also reduce disease in pollinators. Given that parasites have been implicated in ongoing bee declines, this discovery has spurred interest in the potential for 'medicinal' floral products to aid in pollinator conservation efforts. We review the evidence for antiparasitic effects of floral products on bee diseases, emphasizing the importance of investigating the mechanism underlying antiparasitic effects, including direct or host-mediated effects. We discuss the high specificity of antiparasitic effects of even very similar compounds, and highlight the need to consider how nonadditive effects of multiple compounds, and the post-ingestion transformation of metabolites, mediate the disease-reducing capacity of floral products. While the bulk of research on antiparasitic effects of floral products on bee parasites has been conducted in the lab, we review evidence for the impact of such effects in the field, and highlight areas for future research at the floral product-bee disease interface. Such research has great potential both to enhance our understanding of the role of parasites in shaping plant-bee interactions, and the role of plants in determining bee-parasite dynamics. This understanding may in turn reveal new avenues for pollinator conservation.

Interacting Antagonisms: Parasite Infection Alters Bombus impatiens (Hymenoptera: Apidae) Responses to Herbivory on Tomato Plants.

Little is known about how simultaneous antagonistic interactions on plants and pollinators affect pollination services, even though herbivory can alter floral traits and parasites can change pollinator learning, perception, or behavior. We investigated how a common herbivore and bumble bee (Bombus spp.) parasite impact pollination in tomatoes (Solanum lycopersicum L.) (Solanales: Solanaceae). We exposed half the plants to low-intensity herbivory by the specialist Manduca sexta L. (Lepidoptera: Sphigidae), and observed bumble bee visits and time spent on flowers of damaged and control plants. Following observations, we caught the foraging bees and assessed infection by the common gut parasite, Crithidia bombi Lipa & Triggiani (Trypanosomatida: Trypanosomatidae). Interestingly, we found an interactive effect between herbivory and Crithidia infection; bees with higher parasite loads spent less time foraging on damaged plants compared to control plants. However, bees did not visit higher proportions of flowers on damaged or control plants, regardless of infection status. Our study demonstrates that multiple antagonists can have synergistic negative effects on the duration of pollinator visits, such that the consequences of herbivory may depend on the infection status of pollinators. If pollinator parasites indeed exacerbate the negative effects of herbivory on pollination services, this suggests the importance of incorporating bee health management practices to maximize crop production.

Sunflower pollen reduces a gut pathogen in the model bee species, Bombus impatiens, but has weaker effects in three wild congeners.

Commercial bumblebees have become popular models to understand stressors and solutions for pollinator health, but few studies test whether results translate to other pollinators. Consuming sunflower pollen dramatically reduces infection by the gut parasite Crithidia bombi in commercially reared Bombus impatiens. We assessed the effect of sunflower pollen on infection in wild B. impatiens, Bombus griseocollis, Bombus bimaculatus and Bombus vagans. We also asked how pollen diet (50% sunflower pollen versus wildflower pollen) and infection (yes/no) affected performance in wild B. impatiens microcolonies. Compared to controls, sunflower pollen dramatically reduced Crithidia infection in commercial and wild B. impatiens, had similar but less dramatic effects in B. bimaculatus and B. vagans, and no effect in B. griseocollis. Bombus impatiens, B. bimaculatus and B. vagans are in the same subgenus, suggesting that responses to sunflower pollen may be phylogenetically conserved. In microcolonies, 50% sunflower pollen reduced infection compared to wildflower pollen, but also reduced reproduction. Sunflower pollen could control Crithidia infections in B. impatiens and potentially close relatives, but may hinder reproduction if other resources are scarce. We caution that research using managed bee species, such as B. impatiens, be interpreted carefully as findings may not relate to all bee species.

Sunflower pollen induces rapid excretion in bumble bees: Implications for host-pathogen interactions.

Host diet can have a profound effect on host-pathogen interactions, including indirect effects on pathogens mediated through host physiology. In bumble bees (Bombus impatiens), the consumption of sunflower (Helianthus annuus) pollen dramatically reduces infection by the gut protozoan pathogen Crithidia bombi. One hypothesis for the medicinal effect of sunflower pollen is that consumption changes host gut physiological function, causing rapid excretion that flushes C. bombi from the system. We tested the effect of pollen diet and C. bombi infection on gut transit properties using a 2x2 factorial experiment in which bees were infected with C. bombi or not and fed sunflower or wildflower pollen diet. We measured several non-mutually exclusive physiological processes that underlie the insect excretory system, including gut transit time, bi-hourly excretion rate, the total number of excretion events and the total volume of excrement. Sunflower pollen significantly reduced gut transit time in uninfected bees, and increased the total number of excretion events and volume of excrement by 66 % and 68 %, respectively, in both infected and uninfected bees. Here we show that a sunflower pollen diet can affect host physiology gut function, causing more rapid and greater excretion. These results provide important insight into a mechanism that could underlie the medicinal effect of sunflower pollen for bumble bees.

Big bees spread disease: body size mediates transmission of a bumble bee pathogen.

Trait variation can have important consequences for the outcomes of species interactions. Even though some traits vary as much within species as across related species, models and empirical studies typically do not consider the role of intraspecific trait variation for processes such as disease transmission. For example, many pollinator species are in decline because of a variety of stressors including pathogens, but the role of intraspecific trait variation in mediating disease dynamics is rarely considered. For example, pollinator body size could affect pathogen transmission via differences in resistance, foraging behavior and physiology. We tested effects of body size on pollinator pathogen transmission using the common eastern bumble bee Bombus impatiens in field tents, introducing an infected "donor" microcolony of large or small workers with an uninfected average-sized "recipient" microcolony and allowing bees to forage for 9-16 d. Small donor bees had nearly 50% higher infection intensity (cells/0.02  µL) than large donor bees, but large donor bees were twice as likely to transmit Crithidia bombi to recipient bees. Both behavioral and physiological mechanisms may underlie this apparent paradox. Compared to small bees, large bees foraged more and produced more feces; simulations showed that foraging and defecation rates together had stronger effects on transmission than did donor infection intensity. Thus, effects of bee size on contact rates and pathogen supply may play significant roles in disease transmission, demonstrating the multifaceted impacts of traits on transmission dynamics.

Floral traits affecting the transmission of beneficial and pathogenic pollinator-associated microbes.

Flowers provide resources for pollinators, and can also be transmission venues for beneficial or pathogenic pollinator-associated microbes. Floral traits could mediate transmission similarly for beneficial and pathogenic microbes, although some beneficial microbes can grow in flowers while pathogenic microbes may only survive until acquired by a new host. In spite of conceptual similarities, research on beneficial and pathogenic pollinator-associated microbes has progressed mostly independently. Recent advances demonstrate that floral traits are associated with transmission of beneficial and pathogenic microbes, with consequences for pollinator populations and communities. However, there is a near-absence of experimental manipulations of floral traits to determine causal effects on transmission, and a need to understand how floral, microbe and host traits interact to mediate transmission.

Facilitative pollinator sharing decreases with floral similarity in multiple systems.

Investigating the factors that determine whether interactions are competitive or facilitative is essential to understanding community structure and trait evolution. Co-flowering plants interact indirectly through shared pollinators, and meta-analyses suggest that phylogenetic relatedness and floral trait similarity may predict the outcome of these interactions. In a comparative approach, we manipulated the floral community across five focal species to assess how floral similarity and phylogenetic relatedness affect the outcome of interactions. To assess the extent of pollinator-mediated competition versus facilitation, we compared pollen limitation in five focal species growing with floral neighbors (either congeners or neighbors from a different family) relative to a control (growing alone). We measured floral morphology, color, and nectar traits to calculate multivariate floral similarity between species pairs and inferred a phylogeny to calculate phylogenetic distance. Pollinator-mediated interaction values were regressed against floral similarity and phylogenetic distance. We found evidence of pollinator-mediated facilitation in nine of 13 species pairs. Furthermore, floral similarity and phylogenetic distance reduced facilitative interactions, but the latter relationship was not significant when controlling for the identity of the focal species. Our results suggest that facilitative pollinator sharing is more common than reported in the literature, but co-flowering plant species with similar floral traits are less likely to facilitate pollination. A better understanding of the factors that promote facilitation versus competition has important potential applications for managing rare and invasive species.

Colony-Level Effects of Amygdalin on Honeybees and Their Microbes.

Amygdalin, a cyanogenic glycoside, is found in the nectar and pollen of almond trees, as well as in a variety of other crops, such as cherries, nectarines, apples and others. It is inevitable that western honeybees (Apis mellifera) consistently consume amygdalin during almond pollination season because almond crops are almost exclusively pollinated by honeybees. This study tests the effects of a field-relevant concentration of amygdalin on honeybee microbes and the activities of key honeybee genes. We executed a two-month field trial providing sucrose solutions with or without amygdalin ad libitum to free-flying honeybee colonies. We collected adult worker bees at four time points and used RNA sequencing technology and our HoloBee database to assess global changes in microbes and honeybee transcripts. Our hypothesis was that amygdalin will negatively affect bee microbes and possibly immune gene regulation. Using a log2 fold-change cutoff at two and intraday comparisons, we show no large change of bacterial counts, fungal counts or key bee immune gene transcripts, due to amygdalin treatment in relation to the control. However, relatively large titer decreases in the amygdalin treatment relative to the control were found for several viruses. Chronic bee paralysis virus levels had a sharp decrease (-14.4) with titers then remaining less than the control, Black queen cell virus titers were lower at three time points (<-2) and Deformed wing virus titers were lower at two time points (<-6) in amygdalin-fed compared to sucrose-fed colonies. Titers of Lotmaria passim were lower in the treatment group at three of the four dates (<-4). In contrast, Sacbrood virus had two dates with relative increases in its titers (>2). Overall, viral titers appeared to fluctuate more so than bacteria, as observed by highly inconstant patterns between treatment and control and throughout the season. Our results suggest that amygdalin consumption may reduce several honeybee viruses without affecting other microbes or colony-level expression of immune genes.