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Background
VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1.
Results
In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme.
Conclusions
The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
Background:
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.
Results:
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.
Conclusions:
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.
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.
Among the Microbacteriaceae the species of Subtercola and Agreia form closely associated clusters. Phylogenetic analysis demonstrated three major phylogenetic branches of these species. One of these branches contains the two psychrophilic species Subtercola frigoramans and Subtercola vilae, together with a larger number of isolates from various cold environments. Genomic evidence supports the separation of Agreia and Subtercola species. In order to gain insight into the ability of S. vilae to adapt to life in this extreme environment, we analyzed the genome with a particular focus on properties related to possible adaptation to a cold environment. General properties of the genome are presented, including carbon and energy metabolism, as well as secondary metabolite production. The repertoire of genes in the genome of S. vilae DB165\(^T\) linked to adaptations to the harsh conditions found in Llullaillaco Volcano Lake includes several mechanisms to transcribe proteins under low temperatures, such as a high number of tRNAs and cold shock proteins. In addition, S. vilae DB165\(^T\) is capable of producing a number of proteins to cope with oxidative stress, which is of particular relevance at low temperature environments, in which reactive oxygen species are more abundant. Most important, it obtains capacities to produce cryo-protectants, and to combat against ice crystal formation, it produces ice-binding proteins. Two new ice-binding proteins were identified which are unique to S. vilae DB165\(^T\). These results indicate that S. vilae has the capacity to employ different mechanisms to live under the extreme and cold conditions prevalent in Llullaillaco Volcano Lake.
Societal Impact Statement
Pollen relates to many aspects of human and environmental health, which protection and improvement are endorsed by the United Nations Sustainable Development Goals. By highlighting these connections in the frame of current challenges in monitoring and research, we discuss the need of more integrative and multidisciplinary pollen research related to societal needs, improving health of humans and our ecosystems for a sustainable future.
Summary
Pollen is at once intimately part of the reproductive cycle of seed plants and simultaneously highly relevant for the environment (pollinators, vector for nutrients, or organisms), people (food safety and health), and climate (cloud condensation nuclei and climate reconstruction). We provide an interdisciplinary perspective on the many and connected roles of pollen to foster a better integration of the currently disparate fields of pollen research, which would benefit from the sharing of general knowledge, technical advancements, or data processing solutions. We propose a more interdisciplinary and holistic research approach that encompasses total environmental pollen diversity (ePD) (wind and animal and occasionally water distributed pollen) at multiple levels of diversity (genotypic, phenotypic, physiological, chemical, and functional) across space and time. This interdisciplinary approach holds the potential to contribute to pressing human issues, including addressing United Nations Sustainable Development Goals, fostering social and political awareness of these tiny yet important and fascinating particles.
Biodiversity loss, as often found in intensively managed agricultural landscapes, correlates with reduced ecosystem functioning, for example, pollination by insects, and with altered plant composition, diversity, and abundance. But how does this change in floral resource diversity and composition relate to occurrence and resource use patterns of trap-nesting solitary bees? To better understand the impact of land-use intensification on communities of trap-nesting solitary bees in managed grasslands, we investigated their pollen foraging, reproductive fitness, and the nutritional quality of larval food along a land-use intensity gradient in Germany. We found bee species diversity to decrease with increasing land-use intensity irrespective of region-specific community compositions and interaction networks. Land use also strongly affected the diversity and composition of pollen collected by bees. Lack of suitable pollen sources likely explains the absence of several bee species at sites of high land-use intensity. The only species present throughout, Osmia bicornis (red mason bee), foraged on largely different pollen sources across sites. In doing so, it maintained a relatively stable, albeit variable nutritional quality of larval diets (i.e., protein to lipid (P:L) ratio). The observed changes in bee–plant pollen interaction patterns indicate that only the flexible generalists, such as O. bicornis, may be able to compensate the strong alterations in floral resource landscapes and to obtain food of sufficient quality through readily shifting to alternative plant sources. In contrast, other, less flexible, bee species disappear.
Osmotic adaptation and accumulation of compatible solutes is a key process for life at high osmotic pressure and elevated salt concentrations. Most important solutes that can protect cell structures and metabolic processes at high salt concentrations are glycine betaine and ectoine. The genome analysis of more than 130 phototrophic bacteria shows that biosynthesis of glycine betaine is common among marine and halophilic phototrophic Proteobacteria and their chemotrophic relatives, as well as in representatives of Pirellulaceae and Actinobacteria, but are also found in halophilic Cyanobacteria and Chloroherpeton thalassium. This ability correlates well with the successful toleration of extreme salt concentrations. Freshwater bacteria in general lack the possibilities to synthesize and often also to take up these compounds. The biosynthesis of ectoine is found in the phylogenetic lines of phototrophic Alpha- and Gammaproteobacteria, most prominent in the Halorhodospira species and a number of Rhodobacteraceae. It is also common among Streptomycetes and Bacilli. The phylogeny of glycine-sarcosine methyltransferase (GMT) and diaminobutyrate-pyruvate aminotransferase (EctB) sequences correlate well with otherwise established phylogenetic groups. Most significantly, GMT sequences of cyanobacteria form two major phylogenetic branches and the branch of Halorhodospira species is distinct from all other Ectothiorhodospiraceae. A variety of transport systems for osmolytes are present in the studied bacteria.
Bees need food of appropriate nutritional quality to maintain their metabolic functions. They largely obtain all required nutrients from floral resources, i.e., pollen and nectar. However, the diversity, composition and nutritional quality of floral resources varies with the surrounding environment and can be strongly altered in human-impacted habitats. We investigated whether differences in plant species richness as found in the surrounding environment correlated with variation in the floral diversity and nutritional quality of larval provisions (i.e., mixtures of pollen, nectar and salivary secretions) composed by the mass-provisioning stingless bee Tetragonula carbonaria (Apidae: Meliponini). We found that the floral diversity of larval provisions increased with increasing plant species richness. The sucrose and fat (total fatty acid) content and the proportion and concentration of the omega-6 fatty acid linoleic acid decreased, whereas the proportion of the omega-3 fatty acid linolenic acid increased with increasing plant species richness. Protein (total amino acid) content and amino acid composition did not change. The protein to fat (P:F) ratio, known to affect bee foraging, increased on average by more than 40% from plantations to forests and gardens, while the omega-6:3 ratio, known to negatively affect cognitive performance, decreased with increasing plant species richness. Our results suggest that plant species richness may support T. carbonaria colonies by providing not only a continuous resource supply (as shown in a previous study), but also floral resources of high nutritional quality.
The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.
Background
Brood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites’ mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites.
Results
We found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites.
Conclusion
Our results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites.