Nosema ceranae is a microsporidian fungus that parasitizes the midgut epithelial cells of honey bees, Apis mellifera. Due to the role that midgut microorganisms play in bee health and immunity, food supplementation with prebiotics and probiotics may assist in the control of N. ceranae. The dietary fiber prebiotics acacia gum, inulin, and fructooligosaccharides, as well as the commercial probiotics Vetafarm Probotic, Protexin Concentrate single-strain (Enterococcus faecium), and Protexin Concentrate multi-strain (Lactobacillus acidophilus, L. plantarum, L. rhamnosus, L. delbrueckii, Bifidobacterium bifidum, Streptococcus salivarius, and E. faecium) were tested for their effect on N. ceranae spore loads and honey bee survivorship. Bees kept in cages were inoculated with N. ceranae spores and single-dose treatments were administered in sugar syrup. Acacia gum caused the greatest reduction in N. ceranae spore numbers (67%) but also significantly increased bee mortality (62.2%). However, Protexin Concentrate single-strain gave similarly reduced spore numbers (59%) without affecting the mortality. In a second experiment, multiple doses of the probiotics revealed significantly reduced spore numbers with 2.50 mg/mL Vetafarm Probotic, and 0.25, 1.25, and 2.50 mg/mL Protexin Concentrate single-strain. Mortality was also significantly reduced with 1.25 mg/mL Protexin Concentrate single-strain. N. ceranae-inoculated bees fed 3.75 mg/mL Vetafarm Probotic had higher survival than N. ceranae-inoculated bees, which was similar to that of non-inoculated bees, while N. ceranae-inoculated bees fed 2.50 mg/mL Protexin Concentrate single-strain, had significantly higher survival than both N. ceranae-inoculated and non-inoculated bees. Protexin Concentrate single-strain is promising as it can reduce N. ceranae proliferation and increase bee survivorship of infected bees, even compared to healthy, non-infected bees.

Melipona quadrifasciata anthidioides and Scaptotrigona depilis are species of stingless bees capable of producing propolis, which has considerable bioprospecting potential. In this context, the objective of this study was to determine the chemical compositions and evaluate the antimicrobial activity of propolis produced by M. q. anthidioides and S. depilis. The ethanolic extracts of propolis of M. q. anthidioides (EEP-M) and S. depilis (EEP-S) were prepared, and their chemical constituents were characterized by HPLC-ESI-MS. The antimicrobial activity was evaluated against bacteria and fungi, isolated from reference strains and hospital origin resistant to the action of antibiotics. From EEP-M, phenolic compounds were annotated, including gallic acid, ellagic acid, and flavonoids, as well as diterpenes and triterpenes. EEP-S showed mainly triterpene in its chemical composition. Both extracts inhibited the growth of medically relevant bacteria and fungi, including hospital-acquired and antimicrobial-resistant. In general, EEP-S showed better antimicrobial activity compared to EEP-M. The MIC of EEP-S against vancomycin-resistant Enterococcus faecalis was 3.50 mg/mL, while the MIC of EEP-M was 5.33 ± 0.16 mg/mL. In conclusion, this study shows that propolis produced by M. q. anthidioides and S. depilis has the potential to be used for the prevention or treatment of microbial infections.

This study investigated both bacterial and fungal communities in corbicular pollen and hive-stored bee bread of two commercial honey bees, Apis mellifera and Apis cerana, in China. Although both honey bees favor different main floral sources, the dynamics of each microbial community is similar. During pH reduction in hive-stored bee bread, results from conventional culturable methods and next-generation sequencing showed a declining bacterial population but a stable fungal population. Different honey bee species and floral sources might not affect the core microbial community structure but could change the number of bacteria. Corbicular pollen was colonized by the Enterobacteriaceae bacterium (Escherichia-Shiga, Panteoa, Pseudomonas) group; however, the number of bacteria significantly decreased in hive-stored bee bread in less than 72 h. In contrast, Acinetobacter was highly abundant and could utilize protein sources. In terms of the fungal community, the genus Cladosporium remained abundant in both corbicular pollen and hive-stored bee bread. This filamentous fungus might encourage honey bees to reserve pollen by releasing organic acids. Furthermore, several filamentous fungi had the potential to inhibit both commensal/contaminant bacteria and the growth of pathogens. Filamentous fungi, in particular, the genus Cladosporium, could support pollen preservation of both honey bee species.

Honeybees' gut microbiota can provide new valuable access into the pathogenesis-related factors included in infections. Hence, we researched the presence and comparison of gut microbiota groups in control and Nosema spp.-infected honeybee colonies through high-throughput sequencing of the 16S rRNA. As the newest approach in apiary management, we hypothesize that the EM® probiotic for bees could have an important role in therapeutic and immunomodulatory effects on honeybee colonies. The aim of this study was to estimate its impact on the gut microbiota composition of adult honeybees. The major genera were detected, where Lactobacillus was the most abundant genus, followed by Gilliamela, Snodgrassella, and Bifidobacterium. Inoculation with Nosema spp. spores made the relative proportions of Bifidobacterium lower, which was ameliorated by EM® for bees' application. In addition, EM® for bee applied treatments suppressed the increase in the number of Nosema spp. spores. This result points out that continuous EM® for bees treatment shall change bees' gut microbiome composition and mitigate the influence of Nosema spp. infection. Snodgrassella alvi was a major member of the honeybee gut microbiota and may be significantly increased by long-term treatment with EM® for bees. Toward these results, it is possible that EM® for bees treatment will protect honeybees from herbicide glyphosate negative effects in agricultural fields by improving microbiome and immune functions.

Adult honey bees host a remarkably consistent gut microbial community that is thought to benefit host health and provide protection against parasites and pathogens. Currently, however, we lack experimental evidence for the causal role of the gut microbiota in protecting the Western honey bees (Apis mellifera) against their viral pathogens. Here we set out to fill this knowledge gap by investigating how the gut microbiota modulates the virulence of a major honey bee viral pathogen, deformed wing virus (DWV). We found that, upon oral virus exposure, honey bee survival was significantly increased in bees with an experimentally established normal gut microbiota compared to control bees with a perturbed (dysbiotic) gut microbiota. Interestingly, viral titers were similar in bees with normal gut microbiota and dysbiotic bees, pointing to higher viral tolerance in bees with normal gut microbiota. Taken together, our results provide evidence for a positive role of the gut microbiota for honey bee fitness upon viral infection. We hypothesize that environmental stressors altering honey bee gut microbiota composition, e.g., antibiotics in beekeeping or pesticides in modern agriculture, could interact synergistically with pathogens, leading to negative effects on honey bee health and the epidemiology and impact of their viruses.

Pollen stored by bees undergoes a fermentation marked by the presence of lactic acid bacteria and yeasts. It results in bee bread. Past studies have singled out Starmerella (Candida) magnoliae as the most common yeast species in honey bee-stored bee bread. Starmerella species are ecological specialists with potential biotechnological value. The rarity of recent studies on yeasts in honey bees prompted us to generate new information on yeast diversity during the conversion of bee-collected pollen to bee bread. Bees and stored pollen from two apiaries in Belgium were sampled, a yeast isolation protocol was developed, yeast isolates were grouped according to their macro- and micromorphology, and representative isolates were identified using DNA sequences. Most of the 252 identified isolates belonged to the genera Starmerella, Metschnikowia, and Zygosaccharomyces. The high abundance of yeasts in fresh bee bread decreased rapidly with the storage duration. Starmerella species dominated fresh bee bread, while mostly Zygosaccharomyces members were isolated from aged bee bread. Starmerella (Candida) apis, a rarely isolated species, was the most frequent and abundant species in fresh bee bread. Yeasts from the bee's honey stomach and from pollen pellets obtained from bees hind legs were dominated by Metschnikowia species. The distinctive communities from pollen pellets over fresh bee bread to aged bee bread indicate a non-random distribution of these yeasts.

Nosema ceranae (Opisthosporidia: Microsporidia) is an emergent intracellular parasite of the European honey bee (Apis mellifera) and causes serious Nosema disease which has been associated with worldwide honey bee colony losses. The only registered treatment for Nosema disease is fumagillin-b, and this has raised concerns about resistance and off-target effects. Fumagillin-B is banned from use in honey bee colonies in many countries, particularly in Europe. As a result, there is an urgent need for new and effective therapeutic options to treat Nosema disease in honey bees. An RNA interference (RNAi)-based approach can be a potent strategy for controlling diseases in honey bees. We explored the therapeutic potential of silencing the sequences of two N. ceranae encoded spore wall protein (SWP) genes by means of the RNAi-based methodology. Our study revealed that the oral ingestion of dsRNAs corresponding to SWP8 and SWP12 used separately or in combination could lead to a significant reduction in spore load, improve immunity, and extend the lifespan of N. ceranae-infected bees. The results from the work completed here enhance our understanding of honey bee host responses to microsporidia infection and highlight that RNAi-based therapeutics are a promising treatment for honey bee diseases.

Gut microbiota plays important roles in many physiological processes of the host including digestion, protection, detoxification, and development of immune responses. The honey bee (Apis mellifera) has emerged as model for gut-microbiota host interaction studies due to its gut microbiota being highly conserved and having a simple composition. A key gap in this model is understanding how the microbiome differs regionally, including sampling from the tropics and in particular from Africa. The African region is important from the perspective of the native diversity of the bees, and differences in landscape and bee management. Here, we characterized the honey bee gut microbiota in sub-Saharan Africa using 16S rRNA amplicon sequencing. We confirm the presence of the core gut microbiota members and highlight different compositions of these communities across regions. We found that bees from the coastal regions harbor a higher relative abundance and diversity on core members. Additionally, we showed that Gilliamella, Snodgrassella, and Frischella dominate in all locations, and that altitude and humidity affect Gilliamella abundance. In contrast, we found that Lactobacillus was less common compared temperate regions of the world. This study is a first comprehensive characterization of the gut microbiota of honey bees from sub-Saharan Africa and underscores the need to study microbiome diversity in other indigenous bee species and regions.

Nosema ceranae is a widespread parasite responsible for nosemosis Type C in Apis mellifera honey bees, reducing colony survival. The antibiotic fumagillin is the only commercial treatment available, but concerns are emerging about its persistence, safety, and pathogen resistance. The use of natural substances from Brassicaceae defatted seed meals (DSMs) with known antimicrobial and antioxidant properties was explored. Artificially infected bees were fed for 8 days with candies enriched with two concentrations, 2% and 4%, of two DSMs from Brassica nigra and Eruca sativa, containing a known amount of different glucosinolates (GSLs). The food palatability, GSL intake, bee survival, and treatment effects on N. ceranae spore counts were evaluated. Food consumption was higher for the two 2% DSM patties, for both B. nigra and E. sativa, but the GSL intake did not increase by increasing DSM to 4%, due to the resulting lower palatability. The 2% B. nigra patty decreased the bee mortality, while the higher concentration had a toxic effect. The N. ceranae control was significant for all formulates with respect to the untreated control (312,192.6 +/- 14,443.4 s.e.), and was higher for 4% B. nigra (120,366.3 +/- 13,307.1 s.e.). GSL hydrolysis products, the isothiocyanates, were detected and quantified in bee gut tissues. Brassicaceae DSMs showed promising results for their nutraceutical and protective effects on bees artificially infected with N. ceranae spores at the laboratory level. Trials in the field should confirm these results.

Rosenbergiella bacteria have been previously isolated predominantly from floral nectar and identified in metagenomic screenings as associated with bees. Here, we isolated three Rosenbergiella strains from the robust Australian stingless bee Tetragonula carbonaria sharing over 99.4% sequence similarity with Rosenbergiella strains isolated from floral nectar. The three Rosenbergiella strains (D21B, D08K, D15G) from T. carbonaria exhibited near-identical 16S rDNA. The genome of strain D21B was sequenced; its draft genome contains 3,294,717 bp, with a GC content of 47.38%. Genome annotation revealed 3236 protein-coding genes. The genome of D21B differs sufficiently from the closest related strain, Rosenbergiella epipactidis 2.1A, to constitute a new species. In contrast to R. epipactidis 2.1A, strain D21B produces the volatile 2-phenylethanol. The D21B genome contains a polyketide/non-ribosomal peptide gene cluster not present in any other Rosenbergiella draft genomes. Moreover, the Rosenbergiella strains isolated from T. carbonaria grew in a minimal medium without thiamine, but R. epipactidis 2.1A was thiamine-dependent. Strain D21B was named R. meliponini D21B, reflecting its origin from stingless bees. Rosenbergiella strains may contribute to the fitness of T. carbonaria.

Lactic acid bacteria could positively affect the health of honey bees, including nutritional supplementation, immune system development and pathogen colonization resistance. Based on these considerations the present study evaluated predominant Lactic Acid Bacteria (LAB) species from beebread as well as from the social stomach and midgut of Apis mellifera ligustica honey bee foragers. In detail, for each compartment, the diversity in species and biotypes was ascertained through multiple culture-dependent approaches, consisting of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE), 16S rRNA gene sequencing and Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The study of a lactic acid bacteria community, performed with PCR-DGGE and sequence analysis targeting the V1-V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Apilactobacillus kunkeei, Lactiplantibacillus plantarum, Fructobacillus fructosus, Levilactobacillus brevis and Lactobacillus delbrueckii subsp. lactis. Depending on the different compartments, diverse levels of biodiversity in species were found. Particularly, a very low inter-species biodiversity was detected in the midgut that was prevalently dominated by the presence of Apilactobacillus kunkeei. On the other hand, the beebread was characterized by a reasonable biodiversity showing the presence of five species and the predominance of Apilactobacillus kunkeei, Lactiplantibacillus plantarum and Fructobacillus fructosus. The RAPD-PCR analysis performed on the three predominant species allowed the differentiation into several biotypes for each species. Moreover, a relationship between biotypes and compartments has been detected and each biotype was able to express a specific biochemical profile. The biotypes that populated the social stomach and midgut were able to metabolize sugars considered toxic for bees while those isolated from beebread could contribute to release useful compounds with functional properties. Based on this knowledge, new biotechnological approaches could be developed to improve the health of honey bees and the quality of bee products.

Beehives are populated by bacterial species with a protective role against honey bee pathogens thanks to the production of bioactive metabolites. These compounds are largely unexploited despite their high potential interest for pest management. This study evaluated the capability of bacterial species associated with honey bees to produce 2-heptanone, a volatile organic compound with anesthetic properties of the parasitic mite Varroa destructor. The production of this compound was quantified by SPME-GC-MS in a culture filtrate of nine bacterial strains isolated from the surface of honey bees, and the biosynthetic potential was evaluated in bacterial species associated with apiaries by searching for protein homologs putatively involved in its biosynthesis by using biocomputational tools. The findings pointed out that 2-heptanone was produced by Acetobacteraceae bacterium, Bacillus thuringiensis and Apilactobacillus kunkeei isolates in concentrations between 1.5 and 2.6 ng/mL and that its production was strain-specific. Putative methylketone synthase homologs were found in Bacillus, Gilliamella, Acetobacteraceae, Bartonella and Lactobacillaceae, and the protein sequence results were distributed in nine Sequence Similarity Network (SSN) clusters. These preliminary results support the hypothesis that 2-heptanone may act as a mediator of microbial relationships in hives and provide contributions to assess the role and biosynthetic potential of 2-heptanone in apiaries.

The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect's survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees' tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.

Honey bees coexist with fungi that colonize hive surfaces and pollen. Some of these fungi are opportunistic pathogens, but many are beneficial species that produce antimicrobial compounds for pollen conservation and the regulation of pathogen populations. In this study, we tested the in vitro antimicrobial activity of Talaromyces purpureogenus strains isolated from bee bread against Paenibacillus alvei (associated with European foulbrood disease) and three Aspergillus species that cause stonebrood disease. We found that methanol extracts of T. purpureogenus strains B18 and B195 inhibited the growth of P. alvei at a concentration of 0.39 mg/mL. Bioactivity-guided dereplication revealed that the activity of the crude extracts correlated with the presence of diketopiperazines, a siderophore, and three unknown compounds. We propose that non-pathogenic fungi such as Talaromyces spp. and their metabolites in bee bread could be an important requirement to prevent disease. Agricultural practices involving the use of fungicides can disrupt the fungal community and thus negatively affect the health of bee colonies.

Pesticides are increasing honeybee (Apis mellifera) death rates globally. Clothianidin neonicotinoid appears to impair the microbe-immunity axis. We conducted cage experiments on newly emerged bees that were 4-6 days old and used a 16S rRNA metataxonomic approach to measure the impact of three sublethal clothianidin concentrations (0.1, 1 and 10 ppb) on survival, sucrose syrup consumption and gut microbiota community structure. Exposure to clothianidin significantly increased mortality in the three concentrations compared to controls. Interestingly, the lowest clothianidin concentration was associated with the highest mortality, and the medium concentration with the highest food intake. Exposure to clothianidin induced significant variation in the taxonomic distribution of gut microbiota activity. Co-abundance network analysis revealed local dysbiosis signatures specific to each gut section (midgut, ileum and rectum) were driven by specific taxa. Our findings confirm that exposure to clothianidin triggers a reshuffling of beneficial strains and/or potentially pathogenic taxa within the gut, suggesting a honeybee's symbiotic defense systems' disruption, such as resistance to microbial colonization. This study highlights the role of weak transcriptional activity taxa in maintaining a stable honeybee gut microbiota. Finally, the early detection of gut dysbiosis in honeybees is a promising biomarker in hive management for assessing the impact exposure to sublethal xenobiotics.

Honeybees (Apis mellifera L.) are exposed to many parasites, but little is known about interactions with abiotic stressors on their health, particularly when affected as larvae. Larvae were exposed singly and in combination to the parasitic mite Varroa destructor and three sublethal doses of the neonicotinoid insecticide clothianidin to evaluate their effects on survivorship, weight, haemocyte counts, deformed wing virus (DWV) levels and gene expression of the adult bees that subsequently developed. Clothianidin significantly reduced bee weight at the highest dose and was associated with an increase in haemocyte counts at the lowest dose, whereas V. destructor parasitism increased DWV levels, reduced bee emergence, lowered weight and reduced haemocyte counts. An interaction between the two stressors was observed for weight at emergence. Among the differentially expressed genes (DEGs), V. destructor infestation resulted in broader down-regulatory effects related to immunity that was often shared with the combined stressors, while clothianidin resulted in a broader up-regulatory effect more related to central metabolic pathways that was often shared with the combined stressors. Parasites and abiotic stressors can have complex interactions, including additive effects on reduced weight, number of up-regulated DEGs and biological pathways associated with metabolism.

The role of a balanced gut microbiota to maintain health and prevent diseases is largely established in humans and livestock. Conversely, in honeybees, studies on gut microbiota perturbations by external factors have started only recently. Natural methods alternative to chemical products to preserve honeybee health have been proposed, but their effect on the gut microbiota has not been examined in detail. This study aims to investigate the effect of the administration of a bacterial mixture of bifidobacteria and Lactobacillaceae and a commercial product HiveAliveTM on honeybee gut microbiota. The study was developed in 18 hives of about 2500 bees, with six replicates for each experimental condition for a total of three experimental groups. The absolute abundance of main microbial taxa was studied using qPCR and NGS. The results showed that the majority of the administered strains were detected in the gut. On the whole, great perturbations upon the administration of the bacterial mixture and the plant-based commercial product were not observed in the gut microbiota. Significant variations with respect to the untreated control were only observed for Snodgrassella sp. for the bacterial mixture, Bartonella sp. in HiveAliveTM and Bombilactobacillus sp. for both. Therefore, the studied approaches are respectful of the honeybee microbiota composition, conceivably without compromising the bee nutritional, social and ecological functions.

Members of the genus Spiroplasma are Gram-positive bacteria without cell walls. Some Spiroplasma species can cause disease in arthropods such as bees, whereas others provide their host with resistance to pathogens. Ticks also harbour Spiroplasma, but their role has not been elucidated yet. Here, the infection status and genetic diversity of Spiroplasma in ticks were investigated using samples collected from different geographic regions in Japan. A total of 712 ticks were tested for Spiroplasma infection by PCR targeting 16S rDNA, and Spiroplasma species were genetically characterized based on 16S rDNA, ITS, dnaA, and rpoB gene sequences. A total of 109 samples originating from eight tick species were positive for Spiroplasma infection, with infection rates ranging from 0% to 84% depending on the species. A linear mixed model indicated that tick species was the primary factor associated with Spiroplasma infection. Moreover, certain Spiroplasma alleles that are highly adapted to specific tick species may explain the high infection rates in Ixodes ovatus and Haemaphysalis kitaokai. A comparison of the alleles obtained suggests that horizontal transmission between tick species may not be a frequent event. These findings provide clues to understand the transmission cycle of Spiroplasma species in wild tick populations and their roles in host ticks.

Parasites alter the physiology and behaviour of their hosts. In domestic honey bees, the microsporidia Nosema ceranae induces energetic stress that impairs the behaviour of foragers, potentially leading to colony collapse. Whether this parasite similarly affects wild pollinators is little understood because of the low success rates of experimental infection protocols. Here, we present a new approach for infecting bumblebees (Bombus terrestris) with controlled amounts of N. ceranae by briefly exposing individual bumblebees to parasite spores before feeding them with artificial diets. We validated our protocol by testing the effect of two spore dosages and two diets varying in their protein to carbohydrate ratio on the prevalence of the parasite (proportion of PCR-positive bumblebees), the intensity of parasites (spore count in the gut and the faeces), and the survival of bumblebees. Overall, insects fed a low-protein, high-carbohydrate diet showed the highest parasite prevalence (up to 70%) but lived the longest, suggesting that immunity and survival are maximised at different protein to carbohydrate ratios. Spore dosage did not affect parasite infection rate and host survival. The identification of experimental conditions for successfully infecting bumblebees with N. ceranae in the lab will facilitate future investigations of the sub-lethal effects of this parasite on the behaviour and cognition of wild pollinators.

Glyphosate is the most used pesticide around the world. Although different studies have evidenced its negative effect on honey bees, including detrimental impacts on behavior, cognitive, sensory and developmental abilities, its use continues to grow. Recent studies have shown that it also alters the composition of the honey bee gut microbiota. In this study we explored the impact of chronic exposure to sublethal doses of glyphosate on the honey bee gut microbiota and its effects on the immune response, infection by Nosema ceranae and Deformed wing virus (DWV) and honey bee survival. Glyphosate combined with N. ceranae infection altered the structure and composition of the honey bee gut microbiota, for example by decreasing the relative abundance of the core members Snodgrassella alvi and Lactobacillus apis. Glyphosate increased the expression of some immune genes, possibly representing a physiological response to mitigate its negative effects. However, this response was not sufficient to maintain honey bee health, as glyphosate promoted the replication of DWV and decreased the expression of vitellogenin, which were accompanied by a reduced life span. Infection by N. ceranae also alters honey bee immunity although no synergistic effect with glyphosate was observed. These results corroborate previous findings suggesting deleterious effects of widespread use of glyphosate on honey bee health, and they contribute to elucidate the physiological mechanisms underlying a global decline of pollination services.