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.

Contrasting patterns of foraging behavior in neotropical stingless bees using pollen and honey metabarcoding.

Stingless bees are major flower visitors in the tropics, but their foraging preferences and behavior are still poorly understood. Studying stingless bee interactions with angiosperms is methodologically challenging due to the high tropical plant diversity and inaccessibility of upper canopy flowers in forested habitats. Pollen DNA metabarcoding offers an opportunity of assessing floral visitation efficiently and was applied here to understand stingless bee floral resources spectra and foraging behavior. We analyzed pollen and honey from nests of three distantly related stingless bee species, with different body size and social behavior: Melipona rufiventris, Scaptotrigona postica and Tetragonisca angustula. Simultaneously, we evaluate the local floristic components through seventeen rapid botanical surveys conducted at different distances from the nests. We discovered a broad set of explored floral sources, with 46.3 plant species per bee species in honey samples and 53.67 in pollen samples. Plant families Myrtaceae, Asteraceae, Euphorbiaceae, Melastomataceae and Malpighiaceae dominated the records, indicating stingless bee preferences for abundant resources that flowers of these families provide in the region. Results also reinforce the preference of stingless bees for forest trees, even if only available at long distances. Our high-resolution results encourage future bee-plant studies using pollen and honey metabarcoding in hyper-diverse tropical environments.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

Do amino and fatty acid profiles of pollen provisions correlate with bacterial microbiomes in the mason bee Osmia bicornis?

Bee performance and well-being strongly depend on access to sufficient and appropriate resources, in particular pollen and nectar of flowers, which constitute the major basis of bee nutrition. Pollen-derived microbes appear to play an important but still little explored role in the plant pollen-bee interaction dynamics, e.g. through affecting quantities and ratios of important nutrients. To better understand how microbes in pollen collected by bees may affect larval health through nutrition, we investigated correlations between the floral, bacterial and nutritional composition of larval provisions and the gut bacterial communities of the solitary megachilid bee Osmia bicornis. Our study reveals correlations between the nutritional quality of pollen provisions and the complete bacterial community as well as individual members of both pollen provisions and bee guts. In particular pollen fatty acid profiles appear to interact with specific members of the pollen bacterial community, indicating that pollen-derived bacteria may play an important role in fatty acid provisioning. As increasing evidence suggests a strong effect of dietary fatty acids on bee performance, future work should address how the observed interactions between specific fatty acids and the bacterial community in larval provisions relate to health in O. bicornis. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding.

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at - 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

Transitioning the Molecular Tumor Board from Proof of Concept to Clinical Routine: A German Single-Center Analysis.

Molecular precision oncology faces two major challenges: first, to identify relevant and actionable molecular variants in a rapidly changing field and second, to provide access to a broad patient population. Here, we report a four-year experience of the Molecular Tumor Board (MTB) of the Comprehensive Cancer Center Freiburg (Germany) including workflows and process optimizations. This retrospective single-center study includes data on 488 patients enrolled in the MTB from February 2015 through December 2018. Recommendations include individual molecular diagnostics, molecular stratified therapies, assessment of treatment adherence and patient outcomes including overall survival. The majority of MTB patients presented with stage IV oncologic malignancies (90.6%) and underwent an average of 2.1 previous lines of therapy. Individual diagnostic recommendations were given to 487 patients (99.8%). A treatment recommendation was given in 264 of all cases (54.1%) which included a molecularly matched treatment in 212 patients (43.4%). The 264 treatment recommendations were implemented in 76 patients (28.8%). Stable disease was observed in 19 patients (25.0%), 17 had partial response (22.4%) and five showed a complete remission (6.6%). An objective response was achieved in 28.9% of cases with implemented recommendations and for 4.5% of the total population (22 of 488 patients). By optimizing the MTB workflow, case-discussions per session increased significantly while treatment adherence and outcome remained stable over time. Our data demonstrate the feasibility and effectiveness of molecular-guided personalized therapy for cancer patients in a clinical routine setting showing a low but robust and durable disease control rate over time.

Honey bee waggle dance communication increases diversity of pollen diets in intensively managed agricultural landscapes.

The benefits of honey bee dance communication for colony performance in different resource environments are still not well understood. Here, we test the hypothesis that directional dance communication enables honey bee colonies to maintain a diverse pollen diet, especially in landscapes with low resource diversity. To test this hypothesis, we placed 24 Apis mellifera L. colonies with either intact or experimentally disrupted dance communication in eight agricultural landscapes that differed in the diversity of flowering plants and in the dominance of mass-flowering crops. Pollen from incoming foragers was collected and identified via DNA metabarcoding. Disrupting dance communication affected the way the diversity of honey bee pollen diets was impacted by the dominance of mass-flowering crops in available flower resources (p = .04). With increasing dominance of mass-flowering crops in resource environments, foragers of colonies with intact communication foraged on an increasing proportion of available plant genera (p = .01). This was not the case for colonies with disrupted dance communication (p = .5). We conclude that the honey bee dance communication benefits pollen foraging on diverse plant resources and thereby contributes to high quality nutrition in environments with low-resource diversity.

Honey bee foraging ecology: Season but not landscape diversity shapes the amount and diversity of collected pollen.

The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.

Pollen DNA barcoding: current applications and future prospects.

Identification of the species origin of pollen has many applications, including assessment of plant-pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and has few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.

Laboratory rearing of solitary bees and wasps.

Ecological experiments often require standardized methods that exclude natural variation and allow manipulation of a single parameter. It has been shown that domesticated honey bee larvae are raisable in a controlled environment. Here we demonstrate that this approach is also transferable to wild solitary bees and wasps without inducing negative effects on their development. Wells may also be supplemented with the antibiotic substance oxytetracycline to control the presence of bacteria. The method thus provides a useful tool to investigate offspring recruitment and larval development in solitary bees and wasps, plus their responses to manipulation of factors as for example diets, toxins and microbiota.

Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach.

BACKGROUND: Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing. RESULTS: We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000-3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase). CONCLUSIONS: This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.

Diverse microbiota identified in whole intact nest chambers of the red mason bee Osmia bicornis (Linnaeus 1758).

Microbial activity is known to have profound impact on bee ecology and physiology, both by beneficial and pathogenic effects. Most information about such associations is available for colony-building organisms, and especially the honey bee. There, active manipulations through worker bees result in a restricted diversity of microbes present within the colony environment. Microbial diversity in solitary bee nests remains unstudied, although their larvae face a very different situation compared with social bees by growing up in isolated compartments. Here, we assessed the microbiota present in nests and pre-adults of Osmia bicornis, the red mason bee, by culture-independent pyrosequencing. We found high bacterial diversity not comparable with honey bee colonies. We identified a variety of bacteria potentially with positive or negative interactions for bee larvae. However, most of the other diverse bacteria present in the nests seem to originate from environmental sources through incorporated nest building material and stored pollen. This diversity of microorganisms may cause severe larval mortality and require specific physiological or symbiotic adaptations against microbial threats. They may however also profit from such a diverse environment through gain of mutualistic partners. We conclude that further studies of microbiota interaction in solitary bees will improve the understanding of fitness components and populations dynamics.