Search

There is increasing evidence that honeybees (Apis mellifera L.) can adapt naturally to survive Varroa destructor, the primary cause of colony mortality world-wide. Most of the adaptive traits of naturally varroa-surviving honeybees concern varroa reproduction. Here we investigate whether factors in the honeybee metagenome also contribute to this survival. The quantitative and qualitative composition of the bacterial and viral metagenome fluctuated greatly during the active season, but with little overall difference between varroa-surviving and varroa-susceptible colonies. The main exceptions were Bartonella apis and sacbrood virus, particularly during early spring and autumn. Bombella apis was also strongly associated with early and late season, though equally for all colonies. All three affect colony protein management and metabolism. Lake Sinai virus was more abundant in varroa-surviving colonies during the summer. Lake Sinai virus and deformed wing virus also showed a tendency towards seasonal genetic change, but without any distinction between varroa-surviving and varroa-susceptible colonies. Whether the changes in these taxa contribute to survival or reflect demographic differences between the colonies (or both) remains unclear.

The recent worldwide decline of honey bee colonies is a major ecological problem which also threatens pollinated crop production. Several interacting stressors such as environmental pressures and pathogens are suspected. Recently, the gut microbiota has emerged as a critical factor affecting bee health and fitness. We profiled the bacterial communities associated with the gut and whole body of worker bees to assess whether non-thriving colonies could be separated from thriving hives based on their microbial signature. The microbiota of thriving colonies was characterised by higher diversity and higher relative abundance of bacterial taxa involved in sugar degradation that were previously associated with healthy bees (e.g. Commensalibacter sp. and Bartonella apis). In contrast, the microbiota of non-thriving bees was depleted in health-associated species (e.g. Lactobacillus apis), and bacterial taxa associated with disease states (e.g. Gilliamella apicola) and pollen degradation (e.g. G. apicola and Bifidobacterium asteroides) were present in higher abundance compared to thriving colonies. Gut and whole-body microbiota shared a similar dominant core but their comparison showed differences in composition and relative abundance. More differences in taxon relative abundance between gut and whole body were observed in non-thriving bees, suggesting that microbiota associated with other bee organs might also be different. Thus, microbiota profiling could be used as a diagnostic tool in beekeeping practices to predict hive health and guide hive management.

Adult honey bees harbor a specialized gut microbiota of relatively low complexity. While seasonal differences in community composition have been reported, previous studies have focused on compositional changes rather than differences in absolute bacterial loads. Moreover, little is known about the gut microbiota of winter bees, which live much longer than bees during the foraging season, and which are critical for colony survival. We quantified seven core members of the bee gut microbiota in a single colony over two years and characterized the community composition in 14 colonies during summer and winter. Our data shows that total bacterial loads substantially differ between foragers, nurses, and winter bees. Long-lived winter bees had the highest bacterial loads and the lowest community α-diversity, with a characteristic shift towards high levels of Bartonella and Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee experiments, we show that diet is a major contributor to the observed differences in bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is remarkably different from foragers and nurses. Considering the importance of winter bees for colony survival, future work should focus on the role of the gut microbiota in winter bee health and disease.

Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters, and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees ( Apis mellifera) and bumble bees (B ombus sp.). Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella apis, and Serratia strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated knockout of an S. alvi adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities.

Worker honeybees (Apis mellifera) transmit Melissococcus plutonius between colonies. However, the transmission of M. plutonius, which causes European foulbrood (EFB), is poorly understood. To analyze the first EFB outbreak in 40 years in Czechia, we collected 49 hive worker samples from 18 beehives in two diseased apiaries for bacteriome analysis of the V1-V3 portion of the 16S rRNA gene. When we compared control samples obtained outside of the EFB zone, bees from an EFB apiaries containing colonies without clinical symptoms and bees from colonies with EFB clinical symptoms, there was a 100-fold higher occurrence of M. plutonius in colonies with EFB symptoms. The presence of M. plutonius in controls indicated that this pathogen exists in an enzootic state. EFB influenced the core bacteria in the worker bacteriome because the number of Snodgrassella alvi, Lactobacillus mellis, Lactobacillus melliventris, and Fructobacillus fructosus sequences increased, while Bartonella apis, Frischella perrara, and Commensalibacter intestine sequences decreased. Together, the results of this study suggest worker bees from EFB-diseased apiaries serve as vectors of M. plutonius, and eliminating such colonies is an appropriate method to overcome disease outbreaks. Because M. plutonius exists in honeybee colonies in an enzootic state, there may be similar abundances in control colonies outside the EFB zone to those in asymptomatic colonies from EFB apiaries. High-throughput Illumina next-generation sequencing permitted the quantitative interpretation of M. plutonius within the honeybee worker bacteriome. Future studies focusing on honeybee diseases, colony losses, detection of bacterial pathogens and interactions of bacteriome with pathogenic bacteria will benefit of this study.

Background Melissococcus plutonius is an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis mellifera L.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies. Methods The study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1–V3 region, as performed through Illumina MiSeq amplicon sequencing. Results The bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence of M. plutonius than those from EFB1 asymptomatic colonies. Melissococcus plutonius was identified in all EFB1 colonies as well as in some of the control colonies. The proportions of Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Frischella perrara, and Bifidobacterium coryneforme were higher in EFB2 than in EFB1, whereas Lactobacillus mellis was significantly higher in EFB2 than in EFB0. Snodgrassella alvi and L. melliventris, L. helsingborgensis and, L. kullabergensis exhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence of Bartonella apis and Commensalibacter intestini were higher in EFB0 than in EFB2 and EFB1. Enterococcus faecalis incidence was highest in EFB2. Conclusions High-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence of M. plutonius within the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmitting M. plutonius due to the greatly increased incidence of the pathogen. The presence of M. plutonius sequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms that E. faecalis is a secondary invader to M. plutonius; however, other putative secondary invaders were not identified in this study.

The genus Bartonella comprises facultative intracellular bacteria with a unique lifestyle. After transmission by blood-sucking arthropods they colonize the erythrocytes of mammalian hosts causing acute and chronic infectious diseases. Although the pathogen-host interaction is well understood, little is known about the evolutionary origin of the infection strategy manifested by Bartonella species. Here we analyzed six genomes of Bartonella apis, a honey bee gut symbiont that to date represents the closest relative of pathogenic Bartonella species. Comparative genomics revealed that B. apis encodes a large set of vertically inherited genes for amino acid and cofactor biosynthesis and nitrogen metabolism. Most pathogenic bartonellae have lost these ancestral functions, but acquired specific virulence factors and expanded a vertically inherited gene family for harvesting cofactors from the blood. However, the deeply rooted pathogen Bartonella tamiae has retained many of the ancestral genome characteristics reflecting an evolutionary intermediate state toward a host-restricted intraerythrocytic lifestyle. Our findings suggest that the ancestor of the pathogen Bartonella was a gut symbiont of insects and that the adaptation to blood-feeding insects facilitated colonization of the mammalian bloodstream. This study highlights the importance of comparative genomics among pathogens and non-pathogenic relatives to understand disease emergence within an evolutionary-ecological framework.

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the “normal” bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

Here, we report the culture and characterization of an alphaproteobacterium of the order Rhizobiales, isolated from the gut of the honey bee Apis mellifera. Strain PEB0122T shares >95 % 16S rRNA gene sequence similarity with species of the genus Bartonella, a group of mammalian pathogens transmitted by bloodsucking arthropods. Phylogenetic analyses showed that PEB0122T and related strains from the honey bee gut form a sister clade of the genus Bartonella. Optimal growth of strain PEB0122T was obtained on solid media supplemented with defibrinated sheep blood under microaerophilic conditions at 35-37 °C, which is consistent with the cultural characteristics of other species of the genus Bartonella. Reduced growth of strain PEB0122T also occurred under aerobic conditions. The rod-shaped cells of strain PEB0122T had a mean length of 1.2-1.8 μm and revealed hairy surface structures. Strain PEB0122T was positive for catalase, cytochrome c oxidase, urease and nitrate reductase. The fatty acid composition was comparable to those of other species of the genus Bartonella, with palmitic acid (C16 : 0) and isomers of 18- and 19-carbon chains being the most abundant. The genomic DNA G+C content of PEB0122T was determined to be about 45.5 mol%. The high 16S rRNA gene sequence similarity with species of Bartonella and its close phylogenetic position suggest that strain PEB0122T represents a novel species within the genus Bartonella, for which we propose the name Bartonella apis sp. nov. The type strain is PEB0122T ( = NCIMB 14961T = DSM 29779T).

BackgroundMelissococcus plutoniusis an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis melliferaL.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies.MethodsThe study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1–V3 region, as performed through Illumina MiSeq amplicon sequencing.ResultsThe bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence ofM. plutoniusthan those from EFB1 asymptomatic colonies.Melissococcus plutoniuswas identified in all EFB1 colonies as well as in some of the control colonies. The proportions ofFructobacillus fructosus,Lactobacillus kunkeei,Gilliamella apicola,Frischella perrara, andBifidobacterium coryneformewere higher in EFB2 than in EFB1, whereasLactobacillus melliswas significantly higher in EFB2 than in EFB0.Snodgrassella alviandL. melliventris,L. helsingborgensisand,L. kullabergensisexhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence ofBartonella apisandCommensalibacter intestiniwere higher in EFB0 than in EFB2 and EFB1.Enterococcus faecalisincidence was highest in EFB2.ConclusionsHigh-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence ofM. plutoniuswithin the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmittingM. plutoniusdue to the greatly increased incidence of the pathogen. The presence ofM. plutoniussequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms thatE. faecalisis a secondary invader toM. plutonius; however, other putative secondary invaders were not identified in this study.