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

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

Taxonomic distribution of the microbiota according to read classification in both chambers.

<p>Classification is according to the RDP classifier with 0.8 bootstrap cutoff. Reads unassignable at the generic level were included as far as possible in the hierarchical lineage and are displayed with dots. Taxonomic groups with less than 1% share of total number of reads were combined for the sake of clarity and are illustrated by crossed stripes. </p

The bacterial community of the tardigrade Milnesium tardigradum and the effects of antibiotic treatment

<p>Nowadays only little is known about the interaction of tardigrades and their bacteria. Neither the species composition nor the influence of the bacteria onto their hosts have been in focus of researchers. Nevertheless, the high tolerance of tardigrades against environmental stress might be reflected in specialized microbes. Therefore the bacterial community of the tardigrade <em>Milnesium tardigradum</em> from an in-house culture has been investigated. The application of different antibiotic treatment strategies enabled us to alter the microbial distribution. The exact species composition was deciphered by 16S rDNA amplification and sequencing. Using this approach we have established a method to determine the microbiome of tardigrades. Moreover we have been able to identify potential symbiont and food source species in <em>M. tardigradum.</em></p

Classification of Mixed Plant Samples by Next-Generation Sequencing

<p><strong>Classification of Mixed Plant Samples by Next-Generation Sequencing</strong><br> Identification of species in mixed plant samples plays an important role in ecology and sheds light on problems from various research fields. Examples for such samples are pollens (also in bee collections and honey), algae in water samples, food or detritus. The advent of high throughput experiments rendered it possible to obtain sequence data for such samples as an alternative to manual, microscopic classification by experts. But tools for the automated classification of such samples originating from multiple plant species have not been established yet. We developed a bioinformatical workflow to analyze sequences of mixed plant samples through the highly variable species-specific internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA. ITS2 sequences are classified with the naive bayesian RDPclassifier specifically trained with reference sequences from the ITS2 database. To evaluate the performance, we compared results from classical identification based on light microscopy with our sequencing results for 16 bee collected pollen samples. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. The pipeline thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialized expert knowledge and with high throughput.</p

Cryptic species and hidden ecological interactions of halictine bees along an elevational gradient

Abstract Changes of abiotic and biotic conditions along elevational gradients represent serious challenges to organisms which may promote the turnover of species, traits and biotic interaction partners. Here, we used molecular methods to study cuticular hydrocarbon (CHC) profiles, biotic interactions and phylogenetic relationships of halictid bees of the genus Lasioglossum along a 2,900 m elevational gradient at Mt. Kilimanjaro, Tanzania. We detected a strong species turnover of morphologically indistinguishable taxa with phylogenetically clustered cryptic species at high elevations, changes in CHC profiles, pollen resource diversity, and a turnover in the gut and body surface microbiome of bees. At high elevations, increased proportions of saturated compounds in CHC profiles indicate physiological adaptations to prevent desiccation. More specialized diets with higher proportions of low‐quality Asteraceae pollen imply constraints in the availability of food resources. Interactive effects of climatic conditions on gut and surface microbiomes, CHC profiles, and pollen diet suggest complex feedbacks among abiotic conditions, ecological interactions, physiological adaptations, and phylogenetic constraints as drivers of halictid bee communities at Mt. Kilimanjaro

Additional file 2: of Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach

Table S1. Comparison of the number of genera per order for all orders

Standard methods for pollen research

“Bee pollen” is pollen collected from flowers by honey bees. It is used by the bees to nourish themselves, mainly by providing royal jelly and brood food, but it is also used for human nutrition. For the latter purpose, it is collected at the hive entrance as pellets that the bees bring to the hive. Bee pollen has diverse bioactivities, and thus has been used as a health food, and even as medication in some countries. In this paper, we provide standard methods for carrying out research on bee pollen. First, we introduce a method for the production and storage of bee pollen which assures quality of the product. Routine methods are then provided for the identification of the pollen’s floral sources, and determination of the more important quality criteria such as water content and content of proteins, carbohydrates, fatty acids, vitamins, alkaloids, phenolic and polyphenolic compounds. Finally, methods are described for the determination of some important bioactivities of bee pollen such as its antioxidant, anti-inflammatory, antimicrobial and antimutagenic properties. Métodos estándar Para la investigación del polen El "polen de abeja" es el polen recogido de las flores por las abejas melíferas. El polen de abeja es utilizado para nutrir a las propias abejas, principalmente para proporcionar jalea real y alimento para las crías, pero también se utiliza para la nutrición humana. Para este último fin, se recoge en la entrada de la colmena en forma de gránulos que las abejas llevan a la colmena. El polen de abeja tiene diversas bioactividades, por lo que se hautilizado como alimento para la salud, e incluso como medicamento en algunos países. En este artículo, proporcionamos métodos estándar para llevar a cabo investigaciones sobre el polen de abeja. En primer lugar, presentamos un método de producción y almacenamiento de polen de abeja que garantiza la calidad del producto. A continuación, se ofrecen métodos de rutina para la identificación de las fuentes florales del polen y la determinación de los criterios de calidad más importantes, como el contenido de agua y de proteínas, carbohidratos, ácidos grasos, vitaminas, alcaloides y compuestos fenólicos y polifenólicos. Por último, se describen métodos para la determinación de algunas bioactividades importantes del polen de abeja, como sus propiedades antioxidantes, antiinflamatorias, antimicrobianas y antimutagénicas