Bombus Research Articles

Spatiotemporal variability and foraging behavior of bee visitors to a rare long-lived iteroparous forb, Silene spaldingii (Caryophyllaceae).

Silene spaldingii S Watson is a rare long-lived forb (Caryophyllaceae) found primarily in open native grasslands of the Inland Pacific Northwest and is putatively pollinated by one key bumble bee pollinator, Bombus fervidus (Fabricius). However, populations of bumble bees and their visitation patterns can vary dramatically, and some species are in decline including B. fervidus. Understanding the role of co-pollinators such as sweat bees (Halictidae) could be crucial as the plight of rare plants and pollinators intensifies. We collected data across three seasons (2015-2017) on the Bombus-S. spaldingii pollination system, focusing on three Key Conservation Areas in the Channeled Scablands of eastern Washington. Bee visitors to S. spaldingii were monitored and the pool-of-pollinators was surveyed with blue vane traps. Nine species of bees were observed foraging on the plant, while 2211 bees comprised of five families, 22 genera and 81 taxa were captured in blue vane traps, meaning only 11.1% of species in the pollinator pool visited S. spaldingii. Halictus tripartitus Cockerell, a sweat bee, was a common visitor to the plant, but this was the first record of visitation for several other species, including Lasioglossum buccale (Pérez) which has never before been recorded in the Americas. These sweat bees appear to vector S. spaldingii pollen, suggesting they are co-pollinators of the plant. Weather and patch characteristics affected visitation patterns and the pool-of-pollinators. We conclude that sweat bees are likely co-pollinators of S. spaldingii and that they could become increasingly important if B. fervidus populations continue to decline.

Distinct pollinator communities persist among co-flowering specialty crops in Indiana

Diverse bee communities pollinate fruits and vegetables, and the composition of these communities has been described for many specialty crops in major production regions. However, pollinator communities in landscapes dominated by agronomic crops may differ in species composition and the contribution of wild bees. With over 4 million ha of maize and soybean, Indiana presents a novel landscape to compare pollinator communities among specialty crops that differ in their use of managed bees and bloom phenology, with potential implications for the composition of wild bee communities and their contributions to flower visitation. We sampled pollinator communities with flower observations and pan traps in spring-blooming apples and blueberries and summer-blooming tomatoes and watermelons, allowing us to compare communities between specialty crops with overlapping and distinct bloom times. Apples, blueberries, and watermelons were stocked with honey bees, and watermelons additionally had managed bumble bees. Across two years of sampling, we observed 1,651 flower visits by 13 taxa, collected 1,967 bees, and identified 84 taxa from collected specimens. Apples and blueberries hosted the richest pollinator communities (34 and 51 taxa, respectively) followed by watermelons (22 taxa), and tomatoes (19 taxa). While 70% of flower visits in apples and blueberries were attributed to honey bees, wild bees accounted for 82% and 99% of flower visits in watermelons and tomatoes, respectively. Our results indicate that distinct pollinator communities persist among co-flowering specialty crops in a landscape dominated by maize and soybean production and underscore the contribution of wild bees for specialty crop pollination, particularly in watermelons and tomatoes.

Post-transcriptional regulation of Dufour’s gland reproductive signals in bumble bees

Pheromone communication is a key mechanism by which the reproductive division of labor is maintained within insect communities. Understanding how pheromones evolved to regulate social behavior requires knowledge of the molecular regulation of their production. However, even in cases where pheromones were identified, our understanding of their biosynthesis and molecular regulation remains limited. Bumble bees provide a unique system to explore pheromone biosynthesis since workers produce ester sterility signals in their Dufour’s gland that differ from gyne-specific esters and are not produced by queens. These esters are hypothesized to be produced in the exocrine gland where they are stored, and indeed queens, gynes and workers differ significantly in the expression of Dufour’s gland genes coding to enzymes involved in the biosynthesis of esters. However, a previous transcriptome analysis revealed no gene expression differences in the Dufour’s gland of workers despite differences in both ester production and ovarian activation, suggesting that ester production may be regulated lower down. Proteomics of the Dufour’s gland of queens, gynes, and workers recovered over 2400 proteins and broadly matched the previous RNAseq data. However, more than 100 differentially expressed proteins were found between the worker groups, including key enzymes in fatty acid biosynthesis, indicating that the regulation of reproductive signal biosynthesis in workers is done post-transcription. Overall, our data provide evidence that pheromone biosynthesis in the Dufour’s gland is caste specific, that gynes and workers are likely using different enzymes to make their respective wax esters, and that the regulation on pheromone production in queens, gynes and workers is likely done at different regulatory levels, with workers signals being subjected to regulation at the protein level.

A test method for assessing chronic oral toxicity of a pesticide to solitary nesting orchard bees, Osmia spp. (Hymenoptera: Megachilidae).

Orchard bees of the genus Osmia Panzerare important pollinators of fruit trees in various regions of the world, with some species commercially available in the United States and Europe. In addition to their pollination services, Osmia lignaria, Osmia cornifrons, Osmia bicornis, and Osmia cornuta have been identified as potential model species for solitary bees in pesticide risk assessment and have been used for the development of new methods to test acute lethal effects via contact and oral routes of exposure. Our goal was to expand the available methodology to characterize the toxicity of pesticides for these solitary bees through a chronic oral test for adult bees. Chronic oral toxicity of pesticides to orchard bees has been reported, but methods differ among research groups. In our study, O. lignaria, O. cornifrons, O. bicornis, and O. cornuta female bees had access to sucrose solution ad libitum in separate, species-specific 10-day tests. Mean body mass, mean daily consumption, and survival differed among the studied bee species. The dose-response test design was validated with dimethoate, a reference toxic compound, and chronic toxicity endpoints were estimated for the 4 Osmia species. The median lethal daily doses normalized by weight for O. lignaria, O. bicornis, O. cornuta, and O. cornifrons were within the same order of magnitude at 0.23, 0.26, 0.49, and 0.61 µg dimethoate/g bee/day, respectively. The methodology described here was aligned as much as possible with the available honey bee and bumble bee standard methods to facilitate the comparison of chronic toxicity profiles among bee species.

Membership robustness but structural change of the native gut microbiota of bumble bees upon systemic immune induction.

Our work demonstrates how innate immunity may influence the host-associated microbiota. While previous work has demonstrated the role of adaptive immunity in regulating the microbiota, we show that stimulation of an innate immune response in bumble bees may disrupt the native gut microbial community by shifting individual abundances of some members and pairwise associations. This work builds upon previous work in bumble bees demonstrating factors determining microbe colonization of hosts and microbiota membership, implicating immune response-induced changes as a factor shaping these important gut communities. While some microbiota members appear unaffected, changes in others and the community overall suggests that collateral perturbation of the native gut microbiota upon an innate immune response may serve as an additional selective pressure that shapes the evolution of host innate immunity.

Chromosome-scale genome assembly of the hunt bumble bee, Bombus huntii Greene, 1860, a species of agricultural interest.

The Hunt bumble bee, Bombus huntii, is a widely distributed pollinator in western North America. The species produces large colony sizes in captive rearing conditions, experiences low parasite and pathogen loads, and has been demonstrated to be an effective pollinator of tomatoes grown in controlled environment agriculture systems. These desirable traits have galvanized producer efforts to develop commercial Bombus huntii colonies for growers to deliver pollination services to crops. To better understand Bombus huntii biology and support population genetic studies and breeding decisions, we sequenced and assembled the Bombus huntii genome from a single haploid male. High-fidelity sequencing of the entire genome using PacBio, along with HiC sequencing, led to a comprehensive contig assembly of high continuity. This assembly was further organized into a chromosomal arrangement, successfully identifying 18 chromosomes spread across the 317.4 Mb assembly with a BUSCO score indicating 97.6% completeness. Synteny analysis demonstrates shared chromosome number (n = 18) with Bombus terrestris, a species belonging to a different subgenus, matching the expectation that presence of 18 haploid chromosomes is an ancestral trait at least between the subgenera Pyrobombus and Bombus sensu stricto. In conclusion, the assembly outcome, alongside the minimal tissue sampled destructively, showcases efficient techniques for producing a comprehensive, highly contiguous genome.

Sex-Related Variation in Circadian Rhythms in the Bumble Bee Bombus terrestris.

Mating success depends on many factors, but first of all, a male and a female need to meet at the same place and time. The circadian clock is an endogenous system regulating activity and sex-related behaviors in animals. We studied bumble bees (Bombus terrestris) in which the influence of circadian rhythms on sexual behavior has been little explored. We characterized circadian rhythms in adult emergence and locomotor activity under different illumination regimes for males and gynes (unmated queens). We developed a method to monitor adult emergence from the pupal cocoon and found no circadian rhythms in this behavior for either males or gynes. These results are not consistent with the hypothesis that the circadian clock regulates emergence from the pupa in this species. Consistent with this premise, we found that both gynes and males do not show circadian rhythms in locomotor activity during the first 3 days after pupal emergence, but shortly after developed robust circadian rhythms that are readily shifted by a phase delay in illumination regime. We conclude that the bumble bees do not need strong rhythms in adult emergence and during early adult life in their protected and regulated nest environment, but do need strong activity rhythms for timing flights and mating-related behaviors. Next, we tested the hypothesis that the locomotor activity of males and gynes have a similar phase, which may improve mating success. We found that both males and gynes have strong endogenous circadian rhythms that are entrained by the illumination regime, but males show rhythms at an earlier age, their rhythms are stronger, and their phase is slightly advanced relative to that of gynes. An earlier phase may be advantageous to males competing to mate a receptive gyne. Our results are consistent with the hypothesis that sex-related variations in circadian rhythms is shaped by sexual selection.

Learning to handle flowers increases pollen collection for bees but does not affect pollination success for plants

Abstract Cooperation and conflict are common in plant–pollinator interactions. Flowering plants often entice pollinators to visit by offering floral food rewards, thereby facilitating pollination. However, pollinators such as bees can learn to improve their collection of floral rewards (such as pollen), changing how they interact with the flower’s reproductive organs, which together could reduce pollination success. Consequently, complex flowers that slow pollinator learning might benefit the plant. Yet how pollinator learning and flower complexity interact to affect pollination success is unknown. We therefore asked how differences in complexity of 4 flower types (Phacelia campanularia, Exacum affine, Solanum elaeagnifolium, and Erythranthe guttata) affected learning by pollen-foraging generalist bumble bees (Bombus impatiens) and how learning affected pollen collection and pollen deposition on these flowers. We found that bees generally learned how to efficiently handle more complex flower types more slowly. Bees that required more visits to become efficient foragers collected less pollen, with no effect on pollen deposition. Except for the simplest flower type, learning also involved development of motor routines unique to each flower type. Experienced bees overall collected more pollen, but individual differences in motor routines did not affect pollen collection. Conversely, individual differences in motor routines affected pollen deposition, but there was no overall effect of experience. Thus, even though learning overall benefits the bee, it does not alter female (and potentially male) fitness benefits for the plant. We discuss potential reasons for these patterns and consequences for bee behavior and flower evolution.

No impact of cyantraniliprole on the hibernation success of bumble bees (Bombus terrestris audax) in a soil-mediated laboratory exposure study.

Increasing evidence shows that wild bees, including bumble bees, are in decline due to a range of stressors, including pesticides. Our knowledge of pesticide impacts has consequently grown to enable the design of increasingly realistic risk assessment methods. However, one area where knowledge gaps may still hinder our ability to assess the full range of bee-pesticide interactions is the field of exposure. Exposure has historically been linked to either direct contact with pesticides or the ingestion of contaminated pollen and nectar by bees. However, bumble bees, and other wild bees, may also be exposed to pesticides while using contaminated soil as an overwintering substrate. Yet knowledge of how soil-mediated exposure affects bumble bee health is lacking. Here we take one of the first steps towards addressing this knowledge gap by designing a method for testing the effects of soil-mediated pesticide exposure on bumble bee queen hibernation success. We measured hibernation survival, body weight change and abdominal fat content and found that none of these responses were affected by a field realistic soil exposure to the novel insecticide cyantraniliprole. Our study may help in developing a standardised method to test the effects of the soil-mediated pesticide exposure route in bumble bee queens.

The role of foraging pollinators in assembling the flower microbiota and transmitting the fire blight pathogen Erwinia amylovora.

Flowers serve as hubs for biotic interactions with pollinators and microbes, which can significantly impact plant reproduction and health. Previous studies have shown that the flower microbiota undergoes dynamic assembly processes during anthesis. However, the influence of foraging pollinators on the assembly and dispersal of the flower microbiota and the transmission of plant pathogens remains poorly understood. In this study, we used insect exclusion netting to investigate the role of pollinators in the assembly of the microbiota on apple stigma and the transmission of the fire blight pathogen Erwinia amylovora. We found that excluding pollinators had a minor impact on the community diversity and composition of the apple stigma microbiota, while the flower's developmental stage had a strong influence. Additionally, pollinator exclusion altered bacterial dispersal and the relative abundance of different bacterial species, including E. amylovora, suggesting that pollinators play a role in transmitting plant pathogens. Using a reporter system, we demonstrated that bumble bees can transmit the fire blight pathogen from an infected flower under controlled growth conditions. Our study highlights the importance of intrinsic and pollinator-independent microbes as sources of inoculum for the stigma microbiota and underscores the role of foraging pollinators in vectoring plant pathogens.

Testing microbial pest control products in bees, a comparative study on different bee species and their interaction with two representative microorganisms

BackgroundThe evaluation of the impact of pesticides on non-target species, like bees, is a crucial factor in registration procedures. Therefore, standardized test procedures have been developed on OECD level assessing the effects of chemicals on honey bees or bumble bees. Unfortunately, these protocols cannot directly be adapted for testing products that contain microorganisms. Interest in the use of microorganisms has increased in recent years due to their specificity to target species while not harming non-target organisms. This study aimed to evaluate optimal conditions to assess the effects of microbial plant protection products on bee species according to currently available test protocols. Some of the most commonly used microorganisms for plant protection, Bacillus thuringiensis subspecies aizawai (B. t. a. ABTS 1857) and Beauveria bassiana (B. b. ATCC 74040) were tested on Apis mellifera, Bombus terrestris, and Osmia bicornis at different temperatures (18, 26, 33 °C) under laboratory conditions.ResultsExposure to the product containing B. t. a. ABTS 1857 resulted in higher mortality compared to B. b. ATCC 74040 in all tested bee species. A temperature-dependent effect towards higher mortality at higher temperatures of 26 °C or 33 °C was observed in O. bicornis exposed to both microorganisms. A. mellifera showed variable responses, but for B. terrestris there was mostly no effect of temperature when exposed to microorganisms in high concentrations. However, temperature affected longevity of bee species in the non-exposed control group. A. mellifera mortality increased with decreasing temperatures, while B. terrestris and O. bicornis mortality increased with increasing temperatures. A test duration of 15 or 20 days was found to be suitable for testing these microorganisms.ConclusionIn conclusion, 26 °C should be considered the worst-case scenario for testing B. bassiana on all tested bee species. For testing B. thuringiensis, a temperature of 33 °C is recommended for A. mellifera, whereas B. terrestris and O. bicornis should be tested at 26 °C.

The Gut Microbiome of Two Wild Bumble Bee Species Native of South America: Bombus pauloensis and Bombus bellicosus

South America is populated by a wide range of bumble bee species that represent an important source of biodiversity, supporting pollination services in natural and agricultural ecosystems. These pollinators provide unique specific microbial niches, populated by a wide number of microorganisms such as symbionts, environmental opportunistic bacteria, and pathogens. Recently, it was demonstrated how microbial populations are shaped by trophic resources and environmental conditions but also by anthropogenic pressure, which strongly affects microbes’ functionality. This study is focused on the impact of different land uses (natural reserve, agroecosystem, and suburban) on the gut microbiome composition of two South American bumble bees, Bombus pauloensis and Bombus bellicosus. Gut microbial DNA extracted from collected bumble bees was sequenced on the Illumina MiSeq platform and correlated with land use. Nosema ceranae load was analyzed with qPCR and correlated with microbiome data. Significant differences in gut microbiome composition between the two wild bumble bee species were highlighted, with notable variations in α- and β-diversity across study sites. Bombus bellicosus showed a high abundance of Pseudomonas, a genus that includes environmental saprobes, and was found to be the second major taxa populating the gut microbiome, probably indicating the vulnerability of this host to environmental pollution. Pathogen analysis unveils a high prevalence of N. ceranae, with B. bellicosus showing higher susceptibility. Finally, Gilliamella exhibited a negative correlation with N. ceranae, suggesting a potential protective role of this commensal taxon. Our findings underscore the importance of considering microbial dynamics in pollinator conservation strategies, highlighting potential interactions between gut bacteria and pathogens in shaping bumble bee health.

Examining the Effects of Environment, Geography, and Elevation on Patterns of DNA Methylation Across Populations of Two Widespread Bumble Bee Species.

Understanding the myriad avenues through which spatial and environmental factors shape evolution is a major focus in biological research. From a molecular perspective, much work has been focused on genomic sequence variation; however, recently there has been increased interest in how epigenetic variation may be shaped by different variables across the landscape. DNA methylation has been of particular interest given that it is dynamic and can alter gene expression, potentially offering a path for a rapid response to environmental change. We utilized whole genome enzymatic methyl sequencing to evaluate the distribution of CpG methylation across the genome and to analyze patterns of spatial and environmental association in the methylomes of two broadly distributed montane bumble bees (Bombus vancouverensis Cresson and Bombus vosnesenskii Radoszkowski) across elevational gradients in the western US. Methylation patterns in both species are similar at the genomic scale with ∼1% of CpGs being methylated and most methylation being found in exons. At the landscape scale, neither species exhibited strong spatial or population structuring in patterns of methylation, although some weak relationships between methylation and distance or environmental variables were detected. Differential methylation analysis suggests a stronger environment association in B. vancouverensis given the larger number of differentially methylated CpG's compared to B. vosnesenskii. We also observed only a handful of genes with both differentially methylated CpGs and previously detected environmentally associated outlier SNPs. Overall results reveal a weak but present pattern in variation in methylation over the landscape in both species.

Insect Pollinivores of Sorghum bicolor and Plant Traits that Influence Visitation1

Abstract Grasses such as sorghum, (Sorghum bicolor (L.) Moench), are rarely considered as an important food resource for pollinators. Here, we report insects collecting or consuming pollen of sorghum over the 8-wk flowering period of a mapping population in Tifton, GA. We also examine the response of insects to plant morphological traits and disease damage and the ability of bumble bees (Bombus impatiens) to cross-pollinate sorghum. The most numerous insect observed collecting/consuming sorghum pollen was the hover fly Toxomerus politus Say (i.e., the maize calligrapher) followed by honey bees (Apis mellifera L.), lined earwigs (Doru taeniatum Dorhn), lovebugs (Plecia nearctica Hardy), southern carpenter bees (Xylocopa micans Lepeletier), common eastern bumble bees (Bombus impatiens Cresson), exotic stripetails (Allograpta exotica Wiedemann), margined soldier beetles (Chauliognathus marginatus F.), a signal fly (Rivellia sp. Robineau-Desvoidy), and a dusky-winged hover fly (Ocyptamus fuscipennis Say). Maximum flowering occurred at Week 3, which coincided with the maximum number of maize calligraphers, bumble bees, and honey bees observed. A positive linear relationship was seen between the number of flowering plots and the number of maize calligraphers and honey bees. The probability of observing a bee on a panicle increased as plant height increased, whereas the probability of observing a bee or hover fly decreased as plant disease percentage increased. These results suggest that inflorescence abundance, plant height, and plant disease impact bee visitation in sorghum. Furthermore, field-captured bumble bees successfully fertilized sorghum in a greenhouse study, suggesting that bumblebees can pollinate sorghum flowers in field conditions.

Integrative taxonomy of a new species of a bumble bee-mimicking brood parasitic bee, Tetralonioidella mimetica (Hymenoptera, Apoidea, Apidae), investigated through phylogenomics

A new species of bumble bee-mimicking brood parasitic bee, Tetralonioidella mimetica Orr & Zhu, sp. nov., is described from China. The systematic placement of this species was initially challenging but was resolved using a combination of phylogenomic and COI barcode analyses, which strongly support the new species as a member of the genus Tetralonioidella Strand. Interestingly, the new species mimics the color pattern of both a bumble bee (Bombus Latreille), and its host Habropoda Smith species, a mimicry format previously unknown for bees. A review of the other Tetralonioidella species revealed three additional bee mimics, including two further likely model-host-brood parasite mimicry complexes. To our knowledge, these represent the first documented three-tiered mimetic systems in bees. Several additional taxonomic actions recently became necessary in these and related taxa: Tetralonioidella meghalayensis Dohling & Dey, 2024 is synonymized syn. nov. with Habropoda radoszkowskii (Dalla Torre, 1896) and Varthemapistra Engel, stat. rev. is again synonymized with Habrophorula Lieftinck. Our results also highlight issues with the generic classification of the tribe Melectini as currently used, as Melecta Latreille was found paraphyletic in relation to the remaining melectine genera. As a first step toward resolving this issue, we return the Melecta subgenus Eupavlovskia Popov, stat. rev. to genus level and discuss the ongoing systematic uncertainties regarding melectine taxonomy.

Honey bees and bumble bees react differently to nitrogen-induced increases in floral resources

Honey bees and bumble bees react differently to nitrogen-induced increases in floral resources

SolBeePopecotox: A Population Model for Pesticide Risk Assessments of Solitary Bees.

In agricultural landscapes, solitary bees occur in a large diversity of species and are important for crop and wildflower pollination. They are distinguished from honey bees and bumble bees by their solitary lifestyle as well as different nesting strategies, phenologies, and floral preferences. Their ecological traits and presence in agricultural landscapes imply potential exposure to pesticides and suggest a need to conduct ecological risk assessments for solitary bees. However, assessing risks to the large diversity of managed and wild bees across landscapes and regions poses a formidable challenge. Population models provide tools to estimate potential population-level effects of pesticide exposures, can support field study design and interpretation, and can be applied to expand study data to untested conditions. We present a population model for solitary bees, SolBeePopecotox, developed for use in the context of ecological risk assessments. The trait-based model extends a previous version with the explicit representation of exposures to pesticides from relevant routes. Effects are implemented in the model using a simplified toxicokinetic-toxicodynamic model, BeeGUTS (GUTS = generalized unified threshold model for survival), adapted specifically for bees. We evaluated the model with data from semifield studies conducted with the red mason bee, Osmia bicornis, in which bees were foraging in tunnels over control and insecticide-treated oilseed rape fields. We extended the simulations to capture hypothetical semifield studies with two soil-nesting species, Nomia melanderi and Eucera pruinosa, which are difficult to test in empirical studies. The model provides a versatile tool for higher-tier risk assessments, for instance, to estimate effects of potential exposures, expanding available study data to untested species, environmental conditions, or exposure scenarios. Environ Toxicol Chem 2024;00:1-17. © 2024 SETAC.

Bumble bees do not avoid field-realistic but innocuous concentrations of cadmium and copper.

Bee populations are facing numerous stressors globally, including environmental pollution by trace metals and metalloids. Understanding whether bees can detect and avoid these pollutants in their food is pivotal, as avoidance abilities may mitigate their exposure to xenobiotics. While these pollutants are known to induce sublethal effects in bees, such as disrupting physiological mechanisms, their potential impacts on locomotive abilities, fat metabolism, and reproductive physiology remain poorly understood. In this study, utilising workers of the buff-tailed bumble bee and two prevalent trace metals, namely cadmium and copper, we aimed to address these knowledge gaps for field-realistic concentrations. Our findings reveal that workers did not reject field-realistic concentrations of cadmium and copper in sucrose solutions. Moreover, they did not reject lethal concentrations of cadmium, although they rejected lethal concentrations of copper. Additionally, we observed no significant effects of field-realistic concentrations of these metals on the walking and flying activities of workers, nor on their fat metabolism and reproductive physiology. Overall, our results suggest that bumble bees may not avoid cadmium and copper at environmental concentrations, but ingestion of these metals in natural settings may not adversely affect locomotive abilities, fat metabolism, or reproductive physiology. However, given the conservative nature of our study, we still recommend future research to employ higher concentrations over longer durations to mimic conditions in heavily polluted areas (i.e., mine surrounding). Furthermore, investigations should ascertain whether field-realistic concentrations of metals exert no impact on bee larvae.

Stable pollinator communities in different white clover populations suggest potential win-win scenarios for crop yield and biodiversity

Compared to monocultures, intercropping systems offer many agronomic benefits, including higher yield stability. In this study, we assessed whether cropping systems that are beneficial for yield stability are also beneficial for pollinator communities and whether the effect is modulated by the landscape type. Using a replicated block design in one heterogeneous and one homogeneous agricultural landscape, we studied the pollinator communities in eight populations (i.e., genotypes) of white clover (Trifolium repens) grown as a monoculture or as a two-species mixture (together with perennial ryegrass, Lolium perenne) or three-species mixture (together with perennial ryegrass and chicory, Cichorium intybus). We recorded 1486 honey bees and 1254 wild pollinators belonging to 46 species. Bumble bees were the most abundant wild pollinators (49.6 %), followed by hover flies (23.4 %), and non-Bombus wild bees (21.5 %). Lepidoptera accounted for only 5.4 % of the wild pollinators. We found a higher species richness and abundance of wild pollinators in monocultures than in two-species mixtures, but white clover population did not influence pollinators. Moreover, species richness and abundance were also higher in the homogeneous landscape than in the heterogenous one. Most species were foraging on white clover. However, 18 species (39.1 %, n = 18/46) were recorded foraging on chicory and/or weeds, and ten of these wild pollinator species were never recorded on white clover. Our study highlights that diverse pollinator communities require both abundant floral resources and diverse plant communities, that their needs are not in conflict with the goal of achieving yield stability, and that the landscape type can modulate the effect of the cropping system. Moreover, the lack of pollinator preference for different white clover populations suggests that farmers can select mixtures that enhance yield stability without negatively affecting pollinator communities. Overall, these results highlight that intercropping systems comprising several plant species and plant genotypes can guarantee yield stability without compromising the pollinator community, showing that win-win situations for farmers and biodiversity are possible.

The Hole Truth: Why Do Bumble Bees Rob Flowers More Than Once?

Primary nectar-robbers feed through holes they make in flowers, often bypassing the plant's reproductive organs in the process. In many robbed plants, multiple holes are made in a single flower. Why a flower should be robbed repeatedly is difficult to understand: a hole signals that a nectar forager has already fed, which would seem likely to predict low rewards. We tested three explanations for this pattern in Corydalis caseana (Fumariaceae), a bumble bee pollinated and robbed plant: (1) multiple holes appear only after all flowers have been robbed once; (2) individual foragers make multiple holes during single visits; and (3) it is more profitable for bees to rob older flowers, even if they have already been robbed. We tested these hypotheses from 2014 to 2016 in a Colorado, USA population using data on robbing rates over time, floral longevity, nectar accumulation in visited and unvisited flowers, and the accumulation of robbing holes across the life of flowers. Multiple holes were already appearing when two-thirds of flowers still lacked a single hole, allowing us to reject the first hypothesis. The second hypothesis cannot offer a full explanation for multiple robbing holes because 35% of additional holes appeared in flowers one or more days after the first hole was made. Repeated sampling of bagged and exposed inflorescences revealed that flowers filled at a constant rate and refilled completely after being drained. Consequently, young flowers are of consistently low value to foragers compared to older flowers even if they had previously been robbed, consistent with the third hypothesis. While further studies are needed, these results offer a simple explanation for the paradoxical clustering of nectar-robbing damage in this and possibly other plant species.