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).

Despite >130 years of microbial cultivation studies, many microorganisms remain resistant to traditional cultivation approaches, including numerous candidate phyla of bacteria and archaea. Unraveling the mysteries of these candidate phyla is a grand challenge in microbiology and is especially important in habitats where they are abundant, including some extreme environments and low-energy ecosystems. Over the past decade, parallel advances in DNA amplification, DNA sequencing and computing have enabled rapid progress on this problem, particularly through metagenomics and single-cell genomics. Although each approach suffers limitations, metagenomics and single-cell genomics are particularly powerful when combined synergistically. Studies focused on extreme environments have revealed the first substantial genomic information for several candidate phyla, encompassing putative acidophiles (Parvarchaeota), halophiles (Nanohaloarchaeota), thermophiles (Acetothermia, Aigarchaeota, Atribacteria, Calescamantes, Korarchaeota, and Fervidibacteria), and piezophiles (Gracilibacteria). These data have enabled insights into the biology of these organisms, including catabolic and anabolic potential, molecular adaptations to life in extreme environments, unique genomic features such as stop codon reassignments, and predictions about cell ultrastructure. In addition, the rapid expansion of genomic coverage enabled by these studies continues to yield insights into the early diversification of microbial lineages and the relationships within and between the phyla of Bacteria and Archaea. In the next 5 years, the genomic foliage within the tree of life will continue to grow and the study of yet-uncultivated candidate phyla will firmly transition into the post-genomic era.

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.

This account begins with a detailed discussion of historical confusion concerning terminology, and fresh, precise definitions for absconding, migration and swarming are given. Proximate causes for absconding include resource depletion of nectar and pollen and their measurable effects on brood. Predation pressure and microenvironmental effects are discussed as are peculiarities in dance communication in absconding and migration. Finally, comparisons of reproductive swarming and post-absconding behaviour of the species are presented. The relationship between the preparation period for prepared absconding or migration and distance eventually flown is documented as are different foraging strategies for absconding and stationary honeybee colonies. The energetics of absconding/migration in tropical honeybees are considered.

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.

In sexual reproduction, ejaculate components that males transfer into the females vary because of various factors. For male honey bees, the entirety of their sperm is stored in the seminal vesicles until the first copulation with a female and subsequent ejaculation, after which the male will die. Therefore, we can evaluate age-related ejaculate alterations by investigating internal sexual organ changes with age. This study found that seminal vesicle wet weight decreased following sperm transfer from testes to seminal vesicles, while mucus gland weight was unchanged, thus resulting in increased sperm density in the seminal vesicles. This suggests that the decrease of seminal fluid in the seminal vesicles may be the underlying cause for age-related ejaculate component alterations.

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.