Wild bees and their nests host Paenibacillus bacteria with functional potential of avail


In previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.


We collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.


The isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.

Full Text: https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0614-1
by: Alexander Keller, Annette Brandel, Mira C. Becker, Rebecca Balles, Usama Ramadan Abdelmohsen, Markus J. Ankenbrand and Wiebke Sickel

Bacterial community structure and succession in nests of two megachilid bee genera

Studies on honeybees have revealed bacterial taxa which adopt key functions in the hive, in terms of nutrient uptake and immune responses. Despite solitary bees providing invaluable ecological services, the contribution of their microbial communities to larval health and the development and fitness of adults is mainly unknown. To address this gap, we conducted a 16S rDNA meta-barcoding study including larvae and stored pollen in nest chambers from two different megachilid solitary bee genera. We tested how host taxonomy, environmental context and the developmental stage of larvae determined richness and composition of associated bacterial communities. A total of 198 specimens from Osmia bicornis, Osmia caerulescens, Megachile rotundataandMegachile versicolor nests were investigated. Solitary bee bacterial microbiota in the nesting environment were mostly homogeneous within species, and not significantly affected by landscape. For each bee species, we identified bacterial taxa that showed consistent occurrence in the larvae and stored pollen. For the pollen provision, we also described a community shift with progressing larval development, suggesting a reduction of imported floral bacteria.

by: Anna Voulgari-Kokota, Gudrun Grimmer, Ingolf Steffan-Dewenter, Alexander Keller

meta-barcoding marker demultiplexing

A single script! What it does: demultiplexing of metabarcoding data which consists of multiple markers.

  • Data must have followed a library preparation/sequencing strategy which includes sequencing of the forward primers.
  • Data must be demultiplexed for samples already.
  • Sequence data must be in forward orientation.

The categorization is based on Hidden Markov Model (HHMs) hits of the forward primer within the first 20 bp. This is very fast, and allows high throughput of the data.

GitHub: https://github.com/molbiodiv/meta-barcoding-marker-demultiplex

bcgTree: automatized phylogenetic tree building from bacterial core genomes

The need for multi-gene analyses in scientific fields such as phylogenetics and DNA barcoding has increased in recent years. In particular, these approaches are increasingly important for differentiating bacterial species, where reliance on the standard 16S rDNA marker can result in poor resolution. Additionally, the assembly of bacterial genomes has become a standard task due to advances in next-generation sequencing technologies. We created a bioinformatic pipeline, bcgTree, which uses assembled bacterial genomes either from databases or own sequencing results from the user to reconstruct their phylogenetic history. The pipeline automatically extracts 107 essential single-copy core genes, found in a majority of bacteria, using hidden Markov models and performs a partitioned maximum-likelihood analysis.

Freely available at GitHub:  https://github.com/molbiodiv/bcgTree

Publication: http://www.nrcresearchpress.com/doi/abs/10.1139/gen-2015-0175


Pollen/Plant ITS2 reference set for the RDP/UTAX classifier (2015)

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.

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).

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.

Reference: Sickel W, M Ankenbrand, G Grimmer, A Holzschuh,S Härtel, J Lanzen, I Steffan-Dewenter, A Keller (2015) Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecology 15: 20

Github: https://github.com/molbiodiv/meta-barcoding-dual-indexing

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.

Reference: Becker, M., and Keller, A. (2016) Laboratory rearing of solitary bees and wasps, Insect Science 23, 918.



ITS2 database update V (with Dept. of Bioinformatics)

The internal transcribed spacer 2 (ITS2) is a well-established marker for phylogenetic analyses in eukaryotes. A reliable resource for reference sequences and their secondary structures is the ITS2 database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/). However, the database was last updated in 2011. Here, we present a major update of the underlying data almost doubling the number of entities. This increases the number of taxa represented within all major eukaryotic clades. Moreover, additional data has been added to underrepresented groups and some new groups have been added. The broader coverage across the tree of life improves phylogenetic analyses and the capability of ITS2 as a DNA barcode.

Pollen/Plant ITS2 reference set for the RDP classifier (2014)

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, spe- cies-specific internal transcribed spacer two region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database.

To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly cor- related with counted abundances through 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. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

Reference: Keller A, N Danner, G Grimmer, M Ankenbrand, K von der Ohe, W von der Ohe, S Rost, S Härtel, I Steffan-Dewenter (2014) Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples. Plant Biology, 2014

Github: https://github.com/molbiodiv/meta-barcoding-dual-indexing

16S2Genome: Genomic traits for 16S rDNA microbiota studies

Molecular sequencing techniques help to understand microbial biodiversity with regard to species richness, assembly structure and function. In this context, available methods are barcoding, metabarcoding, genomics and metagenomics. The first two are restricted to taxonomic assignments, whilst genomics only refers to functional capabilities of a single organism. Metagenomics by contrast yields information about organismal and functional diversity of a community. However currently it is very demanding regarding labour and costs and thus not applicable to most laboratories. Here, we show in a proof-of-concept that computational approaches are able to retain functional information about microbial communities assessed through 16S rDNA (meta)barcoding by referring to reference genomes. We developed an automatic pipeline to show that such integration may infer preliminary or supplementary genomic content of a community.

Reference: Keller A, Horn H, Förster F, Schultz J. (2014) Computational integration of genomic traits into 16S rDNA microbiota sequencing studies. Gene. 549:1 186–191

Github: https://github.com/molbiodiv/16S2Genome