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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.
The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps .biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full ength and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses.
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.
Solitary bees are subject to a variety of pressures that cause severe population declines. Currently, habitat loss, temperature shifts, agrochemical exposure, and new parasites are identified as major threats. However, knowledge about detrimental bacteria is scarce, although they may disturb natural microbiomes, disturb nest environments, or harm the larvae directly. To address this gap, we investigated 12 Osmia bicornis nests with deceased larvae and 31 nests with healthy larvae from the same localities in a 16S ribosomal RNA (rRNA) gene metabarcoding study. We sampled larvae, pollen provisions, and nest material and then contrasted bacterial community composition and diversity in healthy and deceased nests. Microbiomes of pollen provisions and larvae showed similarities for healthy larvae, whilst this was not the case for deceased individuals. We identified three bacterial taxa assigned to Paenibacillus sp. (closely related to P. pabuli/amylolyticus/xylanexedens), Sporosarcina sp., and Bacillus sp. as indicative for bacterial communities of deceased larvae, as well as Lactobacillus for corresponding pollen provisions. Furthermore, we performed a provisioning experiment, where we fed larvae with untreated and sterilized pollens, as well as sterilized pollens inoculated with a Bacillus sp. isolate from a deceased larva. Untreated larval microbiomes were consistent with that of the pollen provided. Sterilized pollen alone did not lead to acute mortality, while no microbiome was recoverable from the larvae. In the inoculation treatment, we observed that larval microbiomes were dominated by the seeded bacterium, which resulted in enhanced mortality. These results support that larval microbiomes are strongly determined by the pollen provisions. Further, they underline the need for further investigation of the impact of detrimental bacterial acquired via pollens and potential buffering by a diverse pollen provision microbiome in solitary bees.
Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments have demonstrated collective responses of the whole Gr-set. We here disentangle the contributions of all three honeybee sugar receptors (AmGr1-3), combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. We show an expanded sugar spectrum of the AmGr1 receptor. Mutants lacking AmGr1 have a reduced response to sucrose and glucose but not to fructose. AmGr2 solely acts as co-receptor of AmGr1 but not of AmGr3, as we show by electrophysiology and using bimolecular fluorescence complementation. Our results show for the first time that AmGr2 is indeed a functional receptor on its own. Intriguingly, AmGr2 mutants still display a wildtype-like sugar taste. AmGr3 is a specific fructose receptor and is not modulated by a co-receptor. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. Our comprehensive study on the functions of AmGr1, AmGr2 and AmGr3 in honeybees is the first to combine investigations on sugar perception at the receptor level and simultaneously in vivo. We show that honeybees rely on two gustatory receptors to sense all relevant sugars.
The composition and richness of herbivore and plant assemblages change along climatic gradients, but knowledge about associated shifts in specialization is scarce and lacks controlling for the abundance and phylogeny of interaction partners. Thus, we aimed to test whether the specialization of phytophagous insects in insect‐plant interaction networks decreases toward cold habitats as predicted by the ‘altitude niche‐breadth hypothesis’ to forecast possible consequences of interaction rewiring under climate change. We used a non‐invasive, standardized metabarcoding approach to reconstruct dietary relationships of Orthoptera species as a major insect herbivore taxon along a broad temperature gradient (~12°C) in Southern Germany. Based on Orthoptera surveys, feeding observations, collection of fecal pellets from >3,000 individuals of 54 species, and parallel vegetation surveys on 41 grassland sites, we quantified plant resource availability and its use by herbivores. Herbivore assemblages were richer in species and individuals at sites with high summer temperatures, while plant richness peaked at intermediate temperatures. Corresponding interaction networks were most specialized in warm habitats. Considering phylogenetic relationships of plant resources, however, the specialization pattern was not linear but peaked at intermediate temperatures, mediated by herbivores feeding on a narrow range of phylogenetically related resources. Our study provides empirical evidence of resource specialization of insect herbivores along a climatic gradient, demonstrating that resource phylogeny, availability, and temperature interactively shape the specialization of herbivore assemblages. Instead of low specialization levels only in cold, harsh habitats, our results suggest increased generalist feeding due to intraspecific changes and compositional differences at both ends of the microclimatic gradient. We conclude that this nonlinear change of phylogeny‐based resource specialization questions predictions derived from the ‘altitude‐niche breadth hypothesis’ and highlights the currently limited understanding of how plant‐herbivore interactions will change under future climatic conditions.
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.
Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall–specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.
Background: Females have often been shown to exhibit preferences for certain male traits. However, little is known about behavioural rules females use when searching for mates in their natural habitat. We investigated mate sampling tactics and related costs in the territorial strawberry poison frog (Oophaga pumilio) possessing a lek-like mating system, where both sequential and simultaneous sampling might occur. We continuously monitored the sampling pattern and behaviour of females during the complete period between two successive matings.
Results: We found no evidence that females compared males by visiting them. Instead females mated with the closest calling male irrespective of his acoustic and physical traits, and territory size. Playback experiments in the natural home ranges of receptive females revealed that tested females preferred the nearest speaker and did not discriminate between low and high call rates or dominant frequencies.
Conclusions: Our results suggest that females of O. pumilio prefer the closest calling male in the studied population. We hypothesize that the sampling tactic in this population is affected by 1) a strongly female biased sex ratio and 2) a low variance in traits of available males due to strong male-male competition, preventing low quality males from defending a territory and mating.
Tropical peat swamp forests sequester globally significant stores of carbon in deep layers of waterlogged, anoxic, acidic and nutrient-depleted peat. The roles of microbes in supporting these forests through the formation of peat, carbon sequestration and nutrient cycling are virtually unknown. This study investigated physicochemical peat properties and microbial diversity between three dominant tree species: Shorea uliginosa (Dipterocarpaceae), Koompassia malaccensis (legumes associated with nitrogen-fixing bacteria), Eleiodoxa conferta (palm) and depths (surface, 45 and 90 cm) using microbial 16S rRNA gene amplicon sequencing. Water pH, oxygen, nitrogen, phosphorus, total phenolic contents and C/N ratio differed significantly between depths, but not tree species. Depth also strongly influenced microbial diversity and composition, while both depth and tree species exhibited significant impact on the archaeal communities. Microbial diversity was highest at the surface, where fresh leaf litter accumulates, and nutrient supply is guaranteed. Nitrogen was the core parameter correlating to microbial communities, but the interactive effects from various environmental variables displayed significant correlation to relative abundance of major microbial groups. Proteobacteria was the dominant phylum and the most abundant genus, Rhodoplanes, might be involved in nitrogen fixation. The most abundant methanogens and methanotrophs affiliated, respectively, to families Methanomassiliicoccaceae and Methylocystaceae. Our results demonstrated diverse microbial communities and provide valuable insights on microbial ecology in these extreme ecosystems.