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

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.

Linking pollen foraging of megachilid bees to their nest bacterial microbiota.

Solitary bees build their nests by modifying the interior of natural cavities, and they provision them with food by importing collected pollen. As a result, the microbiota of the solitary bee nests may be highly dependent on introduced materials. In order to investigate how the collected pollen is associated with the nest microbiota, we used metabarcoding of the ITS2 rDNA and the 16S rDNA to simultaneously characterize the pollen composition and the bacterial communities of 100 solitary bee nest chambers belonging to seven megachilid species. We found a weak correlation between bacterial and pollen alpha diversity and significant associations between the composition of pollen and that of the nest microbiota, contributing to the understanding of the link between foraging and bacteria acquisition for solitary bees. Since solitary bees cannot establish bacterial transmission routes through eusociality, this link could be essential for obtaining bacterial symbionts for this group of valuable pollinators. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.ebi.ac.uk/ena/data/view/PRJEB27223, https://www.ebi.ac.uk/ena/data/view/PRJEB31610, and https://doi.org/10.5061/dryad.qk36k8q.

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.

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.

Evidence of a novel immune responsive protein in the Hymenoptera.

Honeybee populations are severely threatened by parasites and diseases. Recent outbreaks of Colony Collapse Disorder (CCD) has caused loss of more than 35% of bee colonies in the USA, and this is thought to at least in part be due to parasites and/or disease. Interestingly, the honeybee possesses of a limited set of immune genes compared to other insects. Non-canonical immune genes of honeybee are of interest because they may provide greater insights into the peculiar nature of the immune system of this social insect. Previous analyses of bee haemolymph upon bacterial challenge identified a novel leucine-rich repeat protein termed IRP30. Here we show that IRP30 behaves as a typical secreted immune protein. It is expressed simultaneously with carboxylesterase upon treatment with bacteria or other elicitors of immune response. Furthermore we characterize the gene and the mRNA encoding this protein and the IRP30 protein itself. Its regulation and evolution reveal that IRP30 belongs to a protein family, distributed broadly among Hymenoptera, suggesting its ancient function in immune response. We document an interesting case of a recent IRP30 loss in the ant Atta cephalotes and hypothesize that a putative IRP30 homolog of Nasonia emerged by convergent evolution rather than diverged from a common ancestor.