Single-cell genomics and transcriptomics can provide reliable context for assembled genome fragments and gene expression activity on the level of individual prokaryotic genomes. These methods are rapidly emerging as an essential complement to cultivation-based, metagenomics, metatranscriptomics, and microbial community-focused research approaches by allowing direct access to information from individual microorganisms, even from deep-branching phylogenetic groups that currently lack cultured representatives. Their integration and binning with environmental ‘omics data already provides unprecedented insights into microbial diversity and metabolic potential, enabling us to provide information on individual organisms and the structure and dynamics of natural microbial populations in complex environments. This review highlights the pitfalls and recent advances in the field of single-cell omics and its importance in microbiological and biotechnological studies.

Key points

• Single-cell omics expands the tree of life through the discovery of novel organisms, genes, and metabolic pathways.

• Disadvantages of metagenome-assembled genomes are overcome by single-cell omics.

• Functional analysis of single cells explores the heterogeneity of gene expression.

• Technical challenges still limit this field, thus prompting new method developments.

The complex social structure and male-biased sex ratio of honeybee mating systems are analysed, followed by detailed treatments of panmictic drone congregation areas and species-specific daily mating flight periods. This is followed by an account of queen polyandry and drone monogamy and competition. Mating on the wing is a finely tuned technical tour de force involving initial docking, establishing the internal connection of drone and queen, the deposition and transfer of sperm and, finally, decoupling of the pair and deposition of a mating sign. Subsequent to mating, the problems of the ultimate storage and utilisation of sperm are discussed. Finally, the matter of reproductive isolation is considered.

The bulbus is a part of the unique functional penis (endophallus) in the genus Apis and was studied in Apis mellifera drones. The bulbus consists of a thin cuticular membrane, covered by four different epithelia (dorsal epithelium, anterior epithelium, ventral epithelium I and ventral epithelium II). One day before eclosion, pupae have no sclerotised plates in the bulbus lumen. In emerging drones, however, the lumen contains tanned plate-like structures, the chitin plates, subjacent are small droplets. The chitin plates are connected to each other by a transparent matrix. They consist of globular sclerotized structures which are connected by a network of fibrils. In young adult drones, the dorsal and lateral epithelia consist of highly columnar gland cells which decrease in thickness from the age of 6–12 days from 140 to 20 μm. The ultrastructural analysis combined with histochemistry shows that the gland cells secrete proteinaceous droplets towards the lumen which pass through the cuticle and the pores of the chitin plates and accumulate in the matrix. In sexually mature drones, all secretions have merged to a rigid connective substance, which extends laterally as a thin tapering membrane. The ventral epithelia I and II have no secretory function. The ventral epithelium II has a mucus-like content and several invaginations towards the lumen of the bulbus which enables widening of the bulbus orifice to discharge the bulbus secretions filled with viscous mucus via the cervix into the queen during the mating process.

Aerobic methanotrophs are metabolically unique bacteria that are able to utilize methane as a sole source of energy. They occur in a wide range of habitats where both methane and oxygen are available. Nearly all methanotrophic bacteria that are now isolated in pure cultures belong to the phylum Proteobacteria, while only a limited number of strains represent the Verrucomicrobia. Proteobacterial methanotrophs affiliate with the classes Gammaproteobacteria (type I methanotrophs) and Alphaproteobacteria (type II methanotrophs) and belong to 23 genera and 56 species with validly published names. Several described methanotrophic representatives of the Gammaproteobacteria are not yet obtained in pure cultures and have a “Candidatus” status. Cultivation-independent studies indicate the existence of several further groups of methanotrophic bacteria that have not yet been obtained in culture. However, the currently cultivated strains cover a surprisingly broad range of methanotrophs detected in various environments by molecular approaches. This chapter offers an overview of the currently described methanotroph diversity and the major groups of methanotrophic bacteria that have so far eluded isolation efforts.

Planctomyces brasiliensis Schlesner 1990 belongs to the order Planctomycetales, which differs from other bacterial taxa by several distinctive features such as internal cell compartmentalization, multiplication by forming buds directly from the spherical, ovoid or pear-shaped mother cell and a cell wall consisting of a proteinaceous layer rather than a peptidoglycan layer. The first strains of P. brasiliensis, including the type strain IFAM 1448^T, were isolated from a water sample of Lagoa Vermelha, a salt pit near Rio de Janeiro, Brasil. This is the second completed genome sequence of a type strain of the genus Planctomyces to be published and the sixth type strain genome sequence from the family Planctomycetaceae. The 6,006,602 bp long genome with its 4,811 protein-coding and 54 RNA genes is a part of the GenomicEncyclopedia ofBacteriaandArchaea project. Phylogenomic analyses indicate that the classification within the Planctomycetaceae is partially in conflict with its evolutionary history, as the positioning of Schlesneria renders the genus Planctomyces paraphyletic. A re-analysis of published fatty-acid measurements also does not support the current arrangement of the two genera. A quantitative comparison of phylogenetic and phenotypic aspects indicates that the three Planctomyces species with type strains available in public culture collections should be placed in separate genera. Thus the genera Gimesia, Planctopirus and Rubinisphaera are proposed to accommodate P. maris, P. limnophilus and P. brasiliensis, respectively. Pronounced differences between the reported G + C content of Gemmata obscuriglobus, Singulisphaera acidiphila and Zavarzinella formosa and G + C content calculated from their genome sequences call for emendation of their species descriptions. In addition to other features, the range of G + C values reported for the genera within the Planctomycetaceae indicates that the descriptions of the family and the order should be emended.

In this chapter we will give an overview of international and supranational statistics. The starting point will be the definitions given by Günther Menges [8]: “A statistics is called national if it is — concerning its territorial significance — confined to the territory of a singular legally autonomous state” (p. 14). “A statistics is called international if it consists of more than one national statistics. (…) A (national or international) statistics is called integrated if it consists of the statistics of several (in its statistical meaning) autonomous countries or parts of countries and is in its parts commensurable” (p. 16). “A statistics is called supranational if it is commensurable in its parts and can make holistic statements on the territory of a meaningful set of legally autonomous states. Every supranational statistics should at the same time be considered as integrated and international” (p. 18).

Background

Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.

Methods

Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.

Results

Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.

Conclusions

With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.

Video abstract.

Summary

Queens, homozygous for three marker genes, were inseminated with eight different types of semen, each carrying one combination of the markers. Intracolonial worker relationship (ř_c) was estimated by genotype frequencies in the offspring of the experimental queens. ř_c was larger than under random distribution of semen types in the spermatheca. Estimates of most quantitative genetical parameters will be more accurate using the presented estimator for intracolonial relationship. The insemination order affected genotype frequencies in the offspring, suggesting a weak ‘last male advantage’. There was, however, no evidence of intra spermathecal sperm competition.

Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.