Symbiotic bacteria and fungi proliferate in diapause and may enhance overwintering survival in a solitary bee.

Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here, we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.

Microbes, the 'silent third partners' of bee-angiosperm mutualisms.

While bee-angiosperm mutualisms are widely recognized as foundational partnerships that have shaped the diversity and structure of terrestrial ecosystems, these ancient mutualisms have been underpinned by 'silent third partners': microbes. Here, we propose reframing the canonical bee-angiosperm partnership as a three-way mutualism between bees, microbes, and angiosperms. This new conceptualization casts microbes as active symbionts, processing and protecting pollen-nectar provisions, consolidating nutrients for bee larvae, enhancing floral attractancy, facilitating plant fertilization, and defending bees and plants from pathogens. In exchange, bees and angiosperms provide their microbial associates with food, shelter, and transportation. Such microbial communities represent co-equal partners in tripartite mutualisms with bees and angiosperms, facilitating one of the most important ecological partnerships on land.

Organ-specific volatiles from Sonoran desert Krameria flowers as potential signals for oil-collecting bees.

The evolution of flowers that offer oils as rewards and are pollinated by specialized bees represents a distinctive theme in plant-pollinator co-diversification. Some plants that offer acetylated glycerols as floral oils emit diacetin, a volatile by-product of oil metabolism, which is utilized by oil-collecting bees as an index signal for the presence of floral oil. However, floral oils in the genus Krameria (Krameriaceae) contain ß-acetoxy-substituted fatty acids instead of acetylated glycerols, making them unlikely to emit diacetin as an oil-bee attractant. We analyzed floral headspace composition from K. bicolor and K. erecta, native to the Sonoran Desert of southwestern North America, in search of alternative candidates for volatile index signals. Using solid-phase microextraction, combined with gas chromatography-mass spectrometry, we identified 26 and 45 floral volatiles, respectively, from whole flowers and dissected flower parts of these two Krameria species. As expected, diacetin was not detected. Instead, ß-ionone emerged as a strong candidate for an index signal, as it was uniquely present in dissected oil-producing floral tissues (elaiophores) of K. bicolor, as well as the larval cells and provisions from its oil-bee pollinator, Centris cockerelli. This finding suggests that the floral oil of K. bicolor is perfused with ß-ionone in its tissue of origin and retains the distinctive raspberry-like scent of this volatile after being harvested by C. cockerelli bees. In contrast, the elaiophores of K. erecta, which are not thought to be pollinated by C. cockerelli, produced a blend of anise-related oxygenated aromatics not found in the elaiophores of K. bicolor. Our findings suggest that ß-ionone has the potential to impact oil-foraging by C. cockerelli bees through several potential mechanisms, including larval imprinting on scented provisions or innate or learned preferences by foraging adults.

Individual Variation in Male Pheromone Production in Xylocopa sonorina Correlates with size and Gland Color.

Sex pheromones are species-specific chemical signals that facilitate the location, identification, and selection of mating partners. These pheromones can vary between individuals, and act as signals of mate quality. Here, we investigate the variation of male pheromones in the mesosomal glands of the large carpenter bee Xylocopa sonorina, within a Northern California population. We tested the hypothesis that morphological traits are correlated with the observed variation in chemical blend composition of these bees. We also conducted behavioral assays to test whether these male pheromones act as long-range attractants to conspecifics. We found that larger males with darker mesosomal glands have a higher pheromone amount in their glands. Our analysis also suggests that this pheromone blend functions as a long-range attractant to both males and females. We show that both male body size and sexual maturation are important factors influencing pheromone abundance, and that this pheromone blend acts as a long-range attractant. We hypothesize that this recorded variation in male pheromone could be important for female choice.

Bee breweries: The unusually fermentative, lactobacilli-dominated brood cell microbiomes of cellophane bees.

Pathogens and parasites of solitary bees have been studied for decades, but the microbiome as a whole is poorly understood for most taxa. Comparative analyses of microbiome features such as composition, abundance, and specificity, can shed light on bee ecology and the evolution of host-microbe interactions. Here we study microbiomes of ground-nesting cellophane bees (Colletidae: Diphaglossinae). From a microbial point of view, the diphaglossine genus Ptiloglossa is particularly remarkable: their larval provisions are liquid and smell consistently of fermentation. We sampled larval provisions and various life stages from wild nests of Ptiloglossa arizonensis and two species of closely related genera: Caupolicana yarrowi and Crawfordapis luctuosa. We also sampled nectar collected by P. arizonensis. Using 16S rRNA gene sequencing, we find that larval provisions of all three bee species are near-monocultures of lactobacilli. Nectar communities are more diverse, suggesting ecological filtering. Shotgun metagenomic and phylogenetic data indicate that Ptiloglossa culture multiple species and strains of Apilactobacillus, which circulate among bees and flowers. Larval lactobacilli disappear before pupation, and hence are likely not vertically transmitted, but rather reacquired from flowers as adults. Thus, brood cell microbiomes are qualitatively similar between diphaglossine bees and other solitary bees: lactobacilli-dominated, environmentally acquired, and non-species-specific. However, shotgun metagenomes provide evidence of a shift in bacterial abundance. As compared with several other bee species, Ptiloglossa have much higher ratios of bacterial to plant biomass in larval provisions, matching the unusually fermentative smell of their brood cells. Overall, Ptiloglossa illustrate a path by which hosts can evolve quantitatively novel symbioses: not by acquiring or domesticating novel symbionts, but by altering the microenvironment to favor growth of already widespread and generalist microbes.

Carpenter bee thorax vibration and force generation inform pollen release mechanisms during floral buzzing.

Approximately 10% of flowering plant species conceal their pollen within tube-like poricidal anthers. Bees extract pollen from poricidal anthers via floral buzzing, a behavior during which they apply cyclic forces by biting the anther and rapidly contracting their flight muscles. The success of pollen extraction during floral buzzing relies on the direction and magnitude of the forces applied by the bees, yet these forces and forcing directions have not been previously quantified. In this work, we developed an experiment to simultaneously measure the directional forces and thorax kinematics produced by carpenter bees (Xylocopa californica) during defensive buzzing, a behavior regulated by similar physiological mechanisms as floral buzzing. We found that the buzzing frequencies averaged about 130 Hz and were highly variable within individuals. Force amplitudes were on average 170 mN, but at times reached nearly 500 mN. These forces were 30-80 times greater than the weight of the bees tested. The two largest forces occurred within a plane formed by the bees' flight muscles. Force amplitudes were moderately correlated with thorax displacement, velocity and acceleration amplitudes but only weakly correlated with buzzing frequency. Linear models developed through this work provide a mechanism to estimate forces produced during non-flight behaviors based on thorax kinematic measurements in carpenter bees. Based on the buzzing frequencies, individual bee's capacity to vary buzz frequency and predominant forcing directions, we hypothesize that carpenter bees leverage vibration amplification to increase the deformation of poricidal anthers, and hence the amount of pollen ejected.

Structural dynamics of real and modelled Solanum stamens: implications for pollen ejection by buzzing bees.

An estimated 10% of flowering plant species conceal their pollen within tube-like anthers that dehisce through small apical pores (poricidal anthers). Bees extract pollen from poricidal anthers through a complex motor routine called floral buzzing, whereby the bee applies vibratory forces to the flower stamen by rapidly contracting its flight muscles. The resulting deformation depends on the stamen's natural frequencies and vibration mode shapes, yet for most poricidal species, these properties have not been sufficiently characterized. We performed experimental modal analysis on Solanum elaeagnifolium stamens to quantify their natural frequencies and vibration modes. Based on morphometric and dynamic measurements, we developed a finite-element model of the stamen to identify how variable material properties, geometry and bee weight could affect its dynamics. In general, stamen natural frequencies fell outside the reported floral buzzing range, and variations in stamen geometry and material properties were unlikely to bring natural frequencies within this range. However, inclusion of bee mass reduced natural frequencies to within the floral buzzing frequency range and gave rise to an axial-bending vibration mode. We hypothesize that floral buzzing bees exploit the large vibration amplification factor of this mode to increase anther deformation, which may facilitate pollen ejection.

Neuroanatomical differentiation associated with alternative reproductive tactics in male arid land bees, Centris pallida and Amegilla dawsoni.

Alternative reproductive tactics (ARTs) occur when there is categorical variation in the reproductive strategies of a sex within a population. These different behavioral phenotypes can expose animals to distinct cognitive challenges, which may be addressed through neuroanatomical differentiation. The dramatic phenotypic plasticity underlying ARTs provides a powerful opportunity to study how intraspecific nervous system variation can support distinct cognitive abilities. We hypothesized that conspecific animals pursuing ARTs would exhibit dissimilar brain architecture. Dimorphic males of the bee species Centris pallida and Amegilla dawsoni use alternative mate location strategies that rely primarily on either olfaction (large-morph) or vision (small-morph) to find females. This variation in behavior led us to predict increased volumes of the brain regions supporting their primarily chemosensory or visual mate location strategies. Large-morph males relying mainly on olfaction had relatively larger antennal lobes and relatively smaller optic lobes than small-morph males relying primarily on visual cues. In both species, as relative volumes of the optic lobe increased, the relative volume of the antennal lobe decreased. In addition, A. dawsoni large males had relatively larger mushroom body lips, which process olfactory inputs. Our results suggest that the divergent behavioral strategies in ART systems can be associated with neuroanatomical differentiation.

Does body size predict the buzz-pollination frequencies used by bees?

Body size is an important trait linking pollinators and plants. Morphological matching between pollinators and plants is thought to reinforce pollinator fidelity, as the correct fit ensures that both parties benefit from the interaction. We investigated the influence of body size in a specialized pollination system (buzz-pollination) where bees vibrate flowers to release pollen concealed within poricidal stamens. Specifically, we explored how body size influences the frequency of buzz-pollination vibrations. Body size is expected to affect frequency as a result of the physical constraints it places on the indirect flight muscles that control the production of floral vibrations. Larger insects beat their wings less rapidly than smaller-bodied insects when flying, but whether similar scaling relationships exist with floral vibrations has not been widely explored. This is important because the amount of pollen ejected is determined by the frequency of the vibration and the displacement of a bee's thorax. We conducted a field study in three ecogeographic regions (alpine, desert, grassland) and recorded flight and floral vibrations from freely foraging bees from 27 species across four families. We found that floral vibration frequencies were significantly higher than flight frequencies, but never exceeded 400 Hz. Also, only flight frequencies were negatively correlated with body size. As a bee's size increased, its buzz ratio (floral frequency/flight frequency) increased such that only the largest bees were capable of generating floral vibration frequencies that exceeded double that of their flight vibrations. These results indicate size affects the capacity of bees to raise floral vibration frequencies substantially above flight frequencies. This may put smaller bees at a competitive disadvantage because even at the maximum floral vibration frequency of 400 Hz, their inability to achieve comparable thoracic displacements as larger bees would result in generating vibrations with lower amplitudes, and thus less total pollen ejected for the same foraging effort.

Brawls Bring Buzz: Male Size Influences Competition and Courtship in Diadasia rinconis (Hymenoptera: Apidae).

Sexual selection on male body size in species with a female-biased sexual size dimorphism is common yet often poorly understood. In particular, in the majority of bee species, the relative contribution of intrasexual competition and female choice to patterns of male body size is unknown. In this field study, we examined two possible components of male mating success with respect to body size in the solitary bee Diadasia rinconis Cockerell (Hymenoptera: Apidae): 1) ability to procure a mate and 2) the duration of copulation. We found that larger males were better able to procure mates and copulated for shorter periods of time. Although consistent with sperm competition theory, differences in copulation duration were slight; possibly, the shorter copulations of larger males instead reflect in copulo female choice. Consistent with this notion, males engaged in complex courtship while mounted, characterized for the first time in any bee in such detail via audio recordings and high-speed, high-definition video. The number of pulses in male courtship behavior was also positively associated with copulation duration and may have stimulated females to continue copulating, thereby potentially allowing smaller males to transfer a full ejaculate. Females were shown to be potentially polyandrous and although we did not observe precopulatory rejection in the field, captive females frequently rejected copulation attempts by captive males. Our work indicates that intrasexual competition selects for increased body size in a solitary bee.

The evolution of floral sonication, a pollen foraging behavior used by bees (Anthophila).

Over 22,000 species of biotically pollinated flowering plants, including some major agricultural crops, depend primarily on bees capable of floral sonication for pollination services. The ability to sonicate ("buzz") flowers is widespread in bees but not ubiquitous. Despite the prevalence of this pollinator behavior and its importance to natural and agricultural systems, the evolutionary history of floral sonication in bees has not been previously studied. Here, we reconstruct the evolutionary history of floral sonication in bees by generating a time-calibrated phylogeny and reconstructing ancestral states for this pollen extraction behavior. We also test the hypothesis that the ability to sonicate flowers and thereby efficiently access pollen from a diverse assemblage of plant species, led to increased diversification among sonicating bee taxa. We find that floral sonication evolved on average 45 times within bees, possibly first during the Early Cretaceous (100-145 million years ago) in the common ancestor of bees. We find that sonicating lineages are significantly more species rich than nonsonicating sister lineages when comparing sister clades, but a probabilistic structured rate permutation on phylogenies approach failed to support the hypothesis that floral sonication is a key driver of bee diversification. This study provides the evolutionary framework needed to further study how floral sonication by bees may have facilitated the spread and common evolution of angiosperm species with poricidal floral morphology.

Honeybees (Apis mellifera) learn to discriminate the smell of organic compounds from their respective deuterated isotopomers.

The understanding of physiological and molecular processes underlying the sense of smell has made considerable progress during the past three decades, revealing the cascade of molecular steps that lead to the activation of olfactory receptor (OR) neurons. However, the mode of primary interaction of odorant molecules with the OR proteins within the sensory cells is still enigmatic. Two different concepts try to explain these interactions: the 'odotope hypothesis' suggests that OR proteins recognize structural aspects of the odorant molecule, whereas the 'vibration hypothesis' proposes that intra-molecular vibrations are the basis for the recognition of the odorant by the receptor protein. The vibration hypothesis predicts that OR proteins should be able to discriminate compounds containing deuterium from their common counterparts which contain hydrogen instead of deuterium. This study tests this prediction in honeybees (Apis mellifera) using the proboscis extension reflex learning in a differential conditioning paradigm. Rewarding one odour (e.g. a deuterated compound) with sucrose and not rewarding the respective analogue (e.g. hydrogen-based odorant) shows that honeybees readily learn to discriminate hydrogen-based odorants from their deuterated counterparts and supports the idea that intra-molecular vibrations may contribute to odour discrimination.

Moths on the Flatbed Scanner: The Art of Joseph Scheer.

During the past decade a few artists and even fewer entomologists discovered flatbed scanning technology, using extreme resolution graphical arts scanners for acquiring high magnification digital images of plants, animals and inanimate objects. They are not just for trip receipts anymore. The special attributes of certain scanners, to image thick objects is discussed along with the technical features of the scanners including magnification, color depth and shadow detail. The work of pioneering scanner artist, Joseph Scheer from New York's Alfred University is highlighted. Representative flatbed-scanned images of moths are illustrated along with techniques to produce them. Collecting and preparing moths, and other objects, for scanning are described. Highlights of the Fulbright sabbatical year of professor Scheer in Arizona and Sonora, Mexico are presented, along with comments on moths in science, folklore, art and pop culture. The use of flatbed scanners is offered as a relatively new method for visualizing small objects while acquiring large files for creating archival inkjet prints for display and sale.

Bees assess pollen returns while sonicating Solanum flowers.

Can bees accurately gauge accumulating bodily pollen as they harvest pollen from flowers? Several recent reports conclude that bees fail to assess pollen harvest rates when foraging for nectar and pollen. A native nightshade (Solanum elaeagnifolium Cavanilles) that is visited exclusively for pollen by both solitary and social bees (eg. Ptiloglossa and Bombus) was studied in SE Arizona and SW New Mexico. The flowers have no nectaries. Two experiments were deployed that eliminated "pollen feedback" to the bees by experimentally manipulating flowers prior to bee visits. The two methods were 1) plugging poricidal anthers with glue and 2) emptying anthers of pollen by vibration prior to bee visitation. Both experiments demonstrated that bees directly assess pollen harvest on a flower-by-flower basis, and significantly tailor their handling times, number of vibratile buzzes per flower and grooming bouts according to the ongoing harvest on a given flower. In comparison to experimental flowers, floral handling times were extended for both Bombus and Ptiloglossa on virgin flowers. Greater numbers of intrafloral buzzes and numbers of times bees groomed pollen and packed it into their scopae while still on the flower were also more frequent at virgin versus experimental flowers. Flowers with glued andreocia received uniformly brief visits from Bombus and Ptiloglossa with fewer sonications and virtually no bouts of grooming. Curtailed handling with few buzzes and grooms also characterized visits to our manually harvested flowers wherein pollen was artificially depleted. Sonicating bees respond positively to pollen-feedback while harvesting from individual flowers, and therefore we expect them to adjust their harvesting tempo according to the currency of available pollen (standing crop) within Solanum floral patches.

THE AERODYNAMICS OF POLLEN CAPTURE IN TWO SYMPATRIC EPHEDRA SPECIES.

Wind-tunnel analyses of the behavior of airborne pollen around ovules of two Ephedra species (E. trifurca and E. nevadensis) indicate that at certain airflow speeds (0.5 m/sec and 1.0 m/sec) each species is capable of biasing pollination in favor of conspecific pollen. A computer procedure was designed to evaluate the physical basis for this aerodynamic discrimination. This procedure indicates that differences in size and density confer significantly different inertial properties to the two pollen species. Operating within the specific aerodynamic environments generated around ovules from each species, these differences are sufficient to account for the biases observed in the probability of pollination. Within natural populations, there exists significant variation in pollen size (and possibly in density). Accordingly, it is possible that, under certain ambient wind conditions, ovules from each species can select subsets of the entire airborne population of Ephedra pollen.

Floral biology of the saguaro (Cereus giganteus) : I. Pollen harvest by Apis mellifera.

A saguaro cactus (Cereus giganteus) produces an average of 295 flowers per season, each of which produces 286 mg fresh weight of pollen and 543 mg of nectar containing 24% sugar. At 7600 pollen grains/mg pollen, the yearly output per saguaro plant is 6.4×108 grains. Based on the measured saguaro density of 6.56 plants/ha, 553 g/ha of pollen is produced yearly. The enormous variation among individual plants in terms of flower numbers and floral bloom patterns is documented.Honey bees (Apis mellifera L.), the main collectors of saguaro pollen, collect an average of 12.2 mg pollen per foraging trip and can thus harvest 23.5 pollen loads from one flower. An average honey bee colony collects 290 g of saguaro pollen over the season, which is 24.4% of their total intake. Individual colonies exhibit wide variation in pollen collecting activities with some closely tracking the pollen resource and others almost totally ignoring it. The average for seven colonies indicates that even though variation is great the overall trend is toward closely tracking and exploiting the saguaro pollen resource. Based on the pollen productivity of saguaro and a hypothetical 90% pollen harvesting efficiency of bees, the pollen harvest potential of the saguaro environment is 1.72 colony equivalents of pollen/ha and 0.5/ha for saguaro alone. This is the first quantitative reporting of the total pollen productivity and pollen resource utilization for any plant and an opportunistic pollinator.

Nectar selection by Melipona and Apis mellifera (Hymenoptera: Apidae) and the ecology of nectar intake by bee colonies in a tropical forest.

Colony foraging activity of four Melipona species (Apidae: Meliponinae, tribe Meliponini) was studied during the dry season, when many plants flower in central Panama. The efficiency of sucrose solution uptake by Melipona was compared to that of domesticated European Apis mellifera. Dynamics of nectar foraging were also recorded for 3 of the Melipona visiting the forest shrub, Hybanthus prunifolius (Violaceae). 1. Sugar concentration in nectar brought to nests averaged from 21 to 60% sugar for 15 colonies of M. fasciata, M. compressipes triplaridis, M. fuliginosa and M. marginata micheneri. Concentrations ranged from 19 to 72%, and all species collected nectars ranging at least between 24 and 63% sugar. However, M. compressipes and M. marginata preferred higher concentrations and foraged less on dilute nectars. Peak colony nectar harvest occured in late morning or early afternoon; peak pollen harvest was in early morning. 2. Imbibing rates of bees given 20, 30, 45, 60 or 70% sucrose solutions were highest at ≦45% sucrose, but caloric intake was most rapid at 60% sucrose for all species. All but M. marginata displayed greater net intake rates than domesticated European Apis mellifera. A foraging choice model incorporating caloric reward and imbibing rates of bees suggests M. compressipes and M. marginata should specialize on richer nectars. Rate of caloric intake per forager weight was higher for all Melipona (0.03-0.13g) than for A. mellifera (0.10 g). 3. The nectar of Hybanthus prunifolius (Violaceae), a shrub pollinated exclusively by Melipona, progressed from 35 to 60% sugar during the day. Bees foraged most when nectar was below 60% concentration, a pattern best explained as the result of intercolony competition and greater availability of lower quality nectar. 4. Sugar concentration in nectar harvested by colonies rose from lower to higher values through the day for Melipona. The increasing caloric reward of nectar is adaptive in exploiting foraging preferences of such bees. As standing nectar crop is depleted by competing bees, a gradual shift to more rewarding nectar should promote increased bee foraging range, more flower visits during a foraging trip, floral constancy, and genetic outcrossing. 5. The nectar load capacity of A. mellifera is greater than that of Melipona. Other factors being equal, Africanized A. mellifera, now a permanent resident of neotropical forests, should visit more flowers during a foraging trip. Additional species differences in foraging behavior are analyzed.