Honey Bee Gut Research Articles

Community composition, bacterial symbionts, antibacterial and antioxidant activities of honeybee-associated fungi

BackgroundFungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica remains elusive.ResultsHere, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 μg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 μg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus.ConclusionsThis research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.

Gut Bacterial Diversity and their Antibiotic Resistance in Honey Bee Communities of Dhaka City

The study was undertaken to isolate and identify the bacteria from the gut of honey bees and to investigate their antibiotic sensitivity. A total 35 honey bees were collected from Sher-e-Bangla Agricultural University and Curzon Hall area of Dhaka city. This study was conducted to identify the gram-positive and gram-negative bacteria based on the morphological features using microscope. In this study, four species of honey bees were identified, of which, Apis dorsata was the most dominant (31.43%) species and followed by 17.14% for Apis cerana and 2.86% for Apis mellifera and Apis florea, respectively. In total, five gram-positive bacteria genera including Lactobacillus sp. (37.5%), Streptococcus sp. (9.38%), Staphylococcus sp. (21.88%), Enterococcus sp. (6.25%), and Clostridium sp. (9.38%); and two gram-negative bacteria i.e., Pseudomonas sp. (3.13%), and Enterobacter sp. (12.5%) were detected. Gram-positive bacteria were the most dominant at the honey bee gut in two sites, while bacteria did not detect in A. dorsata gut which was collected from Sher-e-Bangla Agricultural University. However, five types of bacteria were isolated from the gut of A. dorsata collected from Curzon Hall area. In total, the highest varieties (6 out of 7) and number of isolates (15) were found from A. florea. Whereas, the highest number of bacteria was isolated from A. florea (12) for Curzon Hall area, and A. cerana (6) for Sher-e-Bangla Agricultural University. In antibiotic susceptibility test, the isolated Streptococcus sp., Staphylococcus sp. and Enterococcus sp. were sensitive to the majority of the antibiotics. However, the isolated Lactobacilllus sp., Clostridium sp., Enterobacter sp. and Pseudomonas sp. were resistant to the majority of antibiotics. The results show that the presence or absence of bacterium in honey bees gut was an indicator for their overall health. Bangladesh J. Zool. 50 (1): 83-93, 2022

Bifidobacterium mizhiense sp. nov., isolated from the gut of honeybee (Apis mellifera).

A novel bifidobacteria (designated S053-2T) was isolated from the gut of honeybee (Apis mellifera). Strain S053-2T was characterized using a polyphasic taxonomic approach. The result of 16S rRNA gene sequence analysis indicated that strain S053-2T was phylogenetically related to the type strains of Bifidobacterium asteroides, Bifidobacterium indicum, Bifidobacterium actinocoloniiforme, Bifidobacterium xylocopae, Bifidobacterium coryneforme, Bifidobacterium apousia, Bifidobacterium choladohabitans and Bifidobacterium polysaccharolyticum, and had 95.5-99.7 % 16S rRNA gene sequence similarities. Based on the 16S rRNA gene sequence analysis, strain S053-2T was most closely related to the type strain of B. asteroides, having 99.7 % 16S rRNA gene sequence similarity. Strain S053-2T had relatively low (91.6-95.7 %) pheS, atpA, clpC, dnaG, fusA, glnA, glyS, hsp60, argS, pyrG and recA sequence similarities to the type strain of B. asteroides. Strain S053-2T had 94.5-95.3% atpA, clpC, dnaG, dnaK and pyrG sequence similarities to the type strain of B. apousia. The phylogenomic tree indicated that strain S053-2T belonged to the B. asteroides group, and was most closely related to the type strains of B. asteroides, B. apousia, B. choladohabitans and B. polysaccharolyticum, and distantly related to type strains of other phylogenetically related species in the B. asteroides group. Strain S053-2T shared the highest average nucleotide identity (ANI, 93.8 %), digital DNA-DNA hybridization (dDDH, 52.4 %) and average amino acid identity (AAI, 95.6%) values with B. apousia W8102T. Strain S053-2T shared 91.1 % ANI, 41.9 % dDDH and 92.5 % AAI values with B. asteroides DSM 20089T. Acid production from l-arabinose, d-xylose, d-mannose, amygdalin, cellobiose, maltose, melibiose, sucrose, raffinose, gentiobiose and l-fucose, and activity of esterase lipase (C8) and α-fucosidase could differentiate strain S053-2T from B. asteroides DSM 20089T. Acid production from d-mannose, maltose, sucrose, melezitose and gentiobiose, and activity of α-fucosidase could differentiate strain S053-2T from B. apousia W8102T. Based upon the data obtained in the present study, a novel species, Bifidobacterium mizhiense sp. nov., is proposed, and the type strain is S053-2T (=JCM 34710T=CCTCC AB 2021129T).

Apilactobacillus zhangqiuensis sp. nov. and Apilactobacillus xinyiensis sp. nov., isolated from the gut of honeybee (Apis mellifera).

Three Gram-stain-positive bacterial strains (designated F502-1T, F575-4T and F582-1) were isolated from the gut of honeybee (Apis mellifera). These strains were characterized using a polyphasic taxonomic approach. Results of 16S rRNA gene sequence analysis indicated that strain F502-1T was phylogenetically related to the type strains of Apilactobacillus kunkeei, Apilactobacillus nanyangensis and Apilactobacillus apinorum, having 98.7-99.9 % 16S rRNA gene sequence similarities, 84.6-92.5 % pheS sequence similarities and 95.2-99.0 % rpoA sequence similarities, and that strains F575-4T and F582-1 were closely related to the type strain of Apilactobacillus ozensis, having 99.7 and 99.4 % 16S rRNA gene sequence similarities, respectively. Strains F575-4T and F582-1 had less than 88.7 % pheS and 96.4 % rpoA sequence similarities to strain F502-1T and type strains of all Apilactobacillus species. The average nucleotide identity and digital DNA-DNA hybridization values between strains F502-1T, F575-4T, F582-1 and type strains of all Apilactobacillus species were less than 91.3 and 43.5 %, respectively, confirming that they represent two novel species within the genus Apilactobacillus. Based upon the data obtained in the present study, two novel species, Apilactobacillus zhangqiuensis sp. nov. and Apilactobacillus xinyiensis sp. nov., are proposed and the type strains are F502-1T (=JCM 34500T=CCTCC AB 2021026T) and F575-4T (=JCM 34501T=CCTCC AB 2021028T), respectively.

Geographical resistome profiling in the honeybee microbiome reveals resistance gene transfer conferred by mobilizable plasmids

BackgroundThe spread of antibiotic resistance genes (ARGs) has been of global concern as one of the greatest environmental threats. The gut microbiome of animals has been found to be a large reservoir of ARGs, which is also an indicator of the environmental antibiotic spectrum. The conserved microbiota makes the honeybee a tractable and confined ecosystem for studying the maintenance and transfer of ARGs across gut bacteria. Although it has been found that honeybee gut bacteria harbor diverse sets of ARGs, the influences of environmental variables and the mechanism driving their distribution remain unclear.ResultsWe characterized the gut resistome of two closely related honeybee species, Apis cerana and Apis mellifera, domesticated in 14 geographic locations across China. The composition of the ARGs was more associated with host species rather than with geographical distribution, and A. mellifera had a higher content of ARGs in the gut. There was a moderate geographic pattern of resistome distribution, and several core ARG groups were found to be prevalent among A. cerana samples. These shared genes were mainly carried by the honeybee-specific gut members Gilliamella and Snodgrassella. Transferrable ARGs were frequently detected in honeybee guts, and the load was much higher in A. mellifera samples. Genomic loci of the bee gut symbionts containing a streptomycin resistance gene cluster were nearly identical to those of the broad-host-range IncQ plasmid, a proficient DNA delivery system in the environment. By in vitro conjugation experiments, we confirmed that the mobilizable plasmids could be transferred between honeybee gut symbionts by conjugation. Moreover, “satellite plasmids” with fragmented genes were identified in the integrated regions of different symbionts from multiple areas.ConclusionsOur study illustrates that the gut microbiota of different honeybee hosts varied in their antibiotic resistance structure, highlighting the role of the bee microbiome as a potential bioindicator and disseminator of antibiotic resistance. The difference in domestication history is highly influential in the structuring of the bee gut resistome. Notably, the evolution of plasmid-mediated antibiotic resistance is likely to promote the probability of its persistence and dissemination.Aghy2aoGk5W_8F1dgySKf9Video

Social Interaction is Unnecessary for Hindgut Microbiome Transmission in Honey Bees: The Effect of Diet and Social Exposure on Tissue-Specific Microbiome Assembly

Honey bees are a model for host–microbial interactions with experimental designs evolving towards conventionalized worker bees. Research on gut microbiome transmission and assembly has examined only a fraction of factors associated with the colony and hive environment. Here, we studied the effects of diet and social isolation on tissue-specific bacterial and fungal colonization of the midgut and two key hindgut regions. We found that both treatment factors significantly influenced early hindgut colonization explaining similar proportions of microbiome variation. In agreement with previous work, social interaction with older workers was unnecessary for core hindgut bacterial transmission. Exposure to natural eclosion and fresh stored pollen resulted in gut bacterial communities that were taxonomically and structurally equivalent to those produced in the natural colony setting. Stressed diets of no pollen or autoclaved pollen in social isolation resulted in decreased fungal abundance and bacterial diversity, and atypical microbiome structure and tissue-specific variation of functionally important core bacteria. Without exposure to the active hive environment, the abundance and strain diversity of keystone ileum species Gilliamella apicola was markedly reduced. These changes were associated with significantly larger ileum microbiotas suggesting that extended exposure to the active hive environment plays an antibiotic role in hindgut microbiome establishment. We conclude that core hindgut microbiome transmission is facultative horizontal with 5 of 6 core hindgut species readily acquired from the built hive structure and natural diet. Our findings contribute novel insights into factors influencing assembly and maintenance of honey bee gut microbiota and facilitate future experimental designs.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Effect of Chronic Exposure to Sublethal Doses of Imidacloprid and Nosema ceranae on Immunity, Gut Microbiota, and Survival of Africanized Honey Bees.

Large-scale honey bee colony losses reported around the world have been associated with intoxication with pesticides, as with the presence of pests and pathogens. Among pesticides, neonicotinoid insecticides are the biggest threat. Due to their extensive use, they can be found in all agricultural environments, including soil, water, and air, are persistent in the environment, and are highly toxic for honey bees. In addition, infection by different pests and pathogens can act synergistically, weakening bees. In this study, we investigated the effects of chronic exposure to sublethal doses of imidacloprid alone or combined with the microsporidia Nosema ceranae on the immune response, deformed wing virus infection (DWV), gut microbiota, and survival of Africanized honey bees. We found that imidacloprid affected the expression of some genes associated with immunity generating an altered physiological state, although it did not favor DWV or N. ceranae infection. The pesticide alone did not affect honey bee gut microbiota, as previously suggested, but when administered to N. ceranae infected bees, it generated significant changes. Finally, both stress factors caused high mortality rates. Those results illustrate the negative impact of imidacloprid alone or combined with N. ceranae on Africanized honey bees and are useful to understand colony losses in Latin America.

Honeybee gut Lactobacillus modulates host learning and memory behaviors via regulating tryptophan metabolism

Honeybees are highly social insects with a rich behavioral repertoire and are a versatile model for neurobiological research. Their gut microbiota comprises a limited number of host-restricted bacterial phylotypes that are important for honeybee health. However, it remains unclear how specific gut members affect honeybee behaviors. Here, we find that antibiotic exposure disturbs the gut community and influences honeybee phenotypes under field conditions. Using laboratory-generated gnotobiotic bees, we show that a normal gut microbiota is required for olfactory learning and memory abilities. Brain transcriptomic profiling reveals distinct brain gene expression patterns between microbiota-free and conventional bees. Subsequent metabolomic analyses of both hemolymph and gut samples show that the microbiota mainly regulates tryptophan metabolism. Our results indicate that host-specific Lactobacillus strains promote memory behavior by transforming tryptophan to indole derivatives that activate the host aryl hydrocarbon receptor. Our findings highlight the contributions of specific gut members to honeybee neurological processes, thus providing a promising model to understand host-microbe interactions.

Amplicon Sequencing of Single-Copy Protein-Coding Genes Reveals Accurate Diversity for Sequence-Discrete Microbiome Populations.

ABSTRACTAn in-depth understanding of microbial function and the division of ecological niches requires accurate delineation and identification of microbes at a fine taxonomic resolution. Microbial phylotypes are typically defined using a 97% small subunit (16S) rRNA threshold. However, increasing evidence has demonstrated the ubiquitous presence of taxonomic units of distinct functions within phylotypes. These so-called sequence-discrete populations (SDPs) have used to be mainly delineated by disjunct sequence similarity at the whole-genome level. However, gene markers that could accurately identify and quantify SDPs are lacking in microbial community studies. Here, we developed a pipeline to screen single-copy protein-coding genes that could accurately characterize SDP diversity via amplicon sequencing of microbial communities. Fifteen candidate marker genes were evaluated using three criteria (extent of sequence divergence, phylogenetic accuracy, and conservation of primer regions) and the selected genes were subject to test the efficiency in differentiating SDPs within Gilliamella, a core honeybee gut microbial phylotype, as a proof-of-concept. The results showed that the 16S V4 region failed to report accurate SDP diversities due to low taxonomic resolution and changing copy numbers. In contrast, the single-copy genes recommended by our pipeline were able to successfully quantify Gilliamella SDPs for both mock samples and honeybee guts, with results highly consistent with those of metagenomics. The pipeline developed in this study is expected to identify single-copy protein coding genes capable of accurately quantifying diverse bacterial communities at the SDP level.IMPORTANCE Microbial communities can be distinguished by discrete genetic and ecological characteristics. These sequence-discrete populations are foundational for investigating the composition and functional structures of microbial communities at high resolution. In this study, we screened for reliable single-copy protein-coding marker genes to identify sequence-discrete populations through our pipeline. Using marker gene amplicon sequencing, we could accurately and efficiently delineate the population diversity in microbial communities. These results suggest that single copy protein-coding genes can be an accurate, quantitative, and economical alternative for characterizing population diversity. Moreover, the feasibility of a gene as marker for any bacterial population identification can be quickly evaluated by the pipeline proposed here.

Environmental and Anthropogenic Influence on the Core Beneficial Honeybee Gut Microbiota—A Short Communication from Bulgaria

Bees’ and beehives’ health are strongly influenced by the honeybees’ gut microbiota which in turn is strongly dependent on many different factors, including environmental factors as well as anthropogenic pressure. In this study, in four locations in Bulgaria differing strongly in environmental conditions and anthropogenic pressure, an assessment was made using several obligatory core symbiont species and genera for reference, such as Lactobacillus sp., Bifidobacterium sp., Snodgrassiella alvi, Gilliamella apicola, Frishella perrara, and Commensalibacter sp., as well as an observation of the overall number of species. A snapshot of the relative abundances of the total number of species and the core species was made using a next-generation sequencing (NGS)-based metagenomic approach using the Illumina 2 × 250 bp paired-end platform. It was found that the two forms of anthropogenic pressure, the agricultural and the urban/industrial, have distinct effects, affecting different core genera and species. It was also demonstrated that both types of anthropogenic pressure cause a reduction in the overall number of bacterial species.

Correction to: Bioactive potential of invertase by yeast Saccharomyces cerevisiae from the honey bee gut: isolation and characterization

Correction to: Bioactive potential of invertase by yeast Saccharomyces cerevisiae from the honey bee gut: isolation and characterization

Probiotic Potential and Effects on Gut Microbiota Composition and Immunity of Indigenous Gut Lactobacilli in Apis cerana.

This study aimed to investigate the probiotic potential of gut indigenous lactic acid bacteria (LAB) originated from Apis cerana. Six Limosilactobacillus reuteri and one Lactobacillus helveticus were isolated from gut samples of A. cerana adult worker bee. All isolates antagonized the growth of pathogens including Salmonella typhimurium, Escherichia coli, Shigella flexneri, and Flavobacterium frigidimaris, and L. helveticus KM7 showed the greatest antimicrobial activity among them. All strains were sensitive to cefotaxime, amoxicillin, cephalothin, penicillin G, kanamycin, and vancomycin, moderately sensitive to novobiocin and resistant to gentamicin. Six out of seven strains were sensitive to ampicillin. L. helveticus KM7 was chosen to evaluate in vivo probiotic effect of adult worker bees of A. cerana through fed sucrose syrup supplemented with KM7. Administration of KM7 increased survival rate and gut LAB but decreased gut fungi and Enterococcus in honeybees. Expressions of genes related to antimicrobial peptides (AMPs) including Abaecin and Defensin were also induced in the gut of honeybees. The results suggested that L. helveticus KM7 with greater probiotic properties could improve the survival rate of adult worker honeybees of A. cerana through regulating gut microbiota and AMPs genes expression.

Exploring the genome of Lactobacillaceae spp. Sy-1 isolated from Heterotrigona itama honey.

BackgroundHoney produced by Heterotrigona itama is highly preferred among consumers due to its high-value as a functional food and beneficial lactic acid bacteria (LAB) reservoir. Fructophilic lactic acid bacteria (FLAB) are a group of LAB with unique growth characteristics and are regarded as promising producers of bioactive compounds. Hence, it is not surprising that LAB, especially FLAB, may be involved with the excellent bioactivity of H. itama honey. With the trending consumer preference for H. itama honey coupled with increasing awareness for healthy food, the genomic background of FLAB isolated from this honey must, therefore, be clearly understood. In this study, one FLAB strain designated as Sy-1 was isolated from freshly collected H. itama honey. Its FLAB behavior and genomic features were investigated to uncover functional genes that could add value to functional food.MethodsThe fructophilic characteristics of strain Sy-1 were determined, and the genome was sequenced using Illumina iSeq100 and Oxford Nanopore. The average nucleotide identity and phylogenetic analyses based on 16S rRNA, 92 core genes, and whole-genome sequence were performed to unravel the phylogenetic position of strain Sy-1. NCBI Prokaryotic Genome Annotation Pipeline annotated the genome, while the EggNOG-mapper, BLASTKoala, and GHOSTKoala were used to add functional genes and pathways information.ResultsStrain Sy-1 prefers D-fructose over D-glucose and actively metabolizes D-glucose in the presence of electron acceptors. Genomic annotation of strain Sy-1 revealed few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, in line with the characteristic of FLAB. The 16S rRNA gene sequence of strain Sy-1 showed the highest similarity to unknown LAB species isolated from the gut of honeybees. The phylogenetic analyses discovered that strain Sy-1 belonged to the Lactobacillaceae family and formed a separate branch closer to type strain from the genera of Acetilactobacillus and Apilactobacillus. The ANI analysis showed the similarity of the closest relative, Apilactobacillus micheneri Hlig3T. The assembled genome of Sy-1 contains 3 contigs with 2.03 Mbp and a 41% GC content. A total of 1,785 genes were identified, including 1,685 protein-coding genes, 68 tRNA, and 15 rRNA. Interestingly, strain Sy-1 encoded complete genes for the biosynthesis of folate and riboflavin. High-performance liquid chromatography analysis further confirmed the high production of folic acid (1.346 mg/L) by Sy-1.DiscussionBased on phylogenetic and biochemical characteristics, strain Sy-1 should be classified as a novel genus in the family of Lactobacillaceae and a new member of FLAB. The genome information coupled with experimental studies supported the ability of strain Sy-1 to produce high folic acid. Our collective findings support the suitable application of FLAB strain Sy-1 in the functional food and pharmaceutical industries.

Characterization of Bifidobacterium asteroides Isolates.

Bifidobacteria have long been recognized as bacteria with probiotic and therapeutic features. The aim of this work is to characterize the Bifidobacterium asteroides BA15 and BA17 strains, isolated from honeybee gut, to evaluate its safety for human use. An in-depth assessment was carried out on safety properties (antibiotic resistance profiling, β-hemolytic, DNase and gelatinase activities and virulence factor presence) and other properties (antimicrobial activity, auto-aggregation, co-aggregation and hydrophobicity). Based on phenotypic and genotypic characterization, both strains satisfied all the safety requirements. More specifically, genome analysis showed the absence of genes encoding for glycopeptide (vanA, vanB, vanC-1, vanC-2, vanD, vanE, vanG), resistance to tetracycline (tetM, tetL and tetO) and virulence genes (asa1, gelE, cylA, esp, hyl).

Distinct Roles of Honeybee Gut Bacteria on Host Metabolism and Neurological Processes.

ABSTRACTThe honeybee possesses a limited number of bacterial phylotypes that play essential roles in host metabolism, hormonal signaling, and feeding behavior. However, the contribution of individual gut members in shaping honeybee brain profiles remains unclear. By generating gnotobiotic bees which were mono-colonized by a single gut bacterium, we revealed that different species regulated specific modules of metabolites in the hemolymph. Circulating metabolites involved in carbohydrate and glycerophospholipid metabolism pathways were mostly regulated by Gilliamella, while Lactobacillus Firm4 and Firm5 mainly altered amino acid metabolism pathways. We then analyzed the brain transcriptomes of bees mono-colonized with these three bacteria. These showed distinctive gene expression profiles, and genes related to olfactory functions and labor division were upregulated by Lactobacillus. Interestingly, differentially spliced genes in the brains of gnotobiotic bees largely overlapped with those of bees unresponsive to social stimuli. The differentially spliced genes were enriched in pathways involved in neural development and synaptic transmission. We showed that gut bacteria altered neurotransmitter levels in the brain. In particular, dopamine and serotonin, which show inhibitory effects on the sensory sensitivity of bees, were downregulated in bacteria-colonized bees. The proboscis extension response showed that a normal gut microbiota is essential for the taste-related behavior of honeybees, suggesting the contribution of potential interactions among different gut species to the host’s physiology. Our findings provide fundamental insights into the diverse functions of gut bacteria which likely contribute to honeybee neurological processes.IMPORTANCE The honeybee possesses a simple and host-restricted gut community that contributes to the metabolic health of its host, while the effects of bacterial symbionts on host neural functions remain elusive. We found that the colonization of specific bee gut bacteria regulates distinct circulating metabolites enriched in carbohydrate, amino acid, and glycerophospholipid metabolic pathways. The brains of bees colonized with different gut members display distinct transcriptomic profiles of genes crucial for bee behaviors and division of labor. Alternative splicing of genes related to disordered bee behaviors is also mediated. The presence of gut bacteria promotes sucrose sensitivity with major neurotransmitters being regulated in the brain. Our findings demonstrate how individual bee gut species affect host behaviors, highlighting the gut-brain connections important for honeybee neurobiological and physiological states.

Enzymatic and structural characterization of β-fructofuranosidase from the honeybee gut bacterium Frischella perrara.

Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF2), tetrasaccharide nystose (GF3), and fructosyl nystose (GF4). Enzymes that hydrolyze GF3 may be useful for preparing GF2 from the fructooligosaccharide mixture. A β-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60mM sucrose, GF2, and GF3 by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF2 was lower than those for disaccharide sucrose and tetrasaccharide GF3. The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial β-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF2 and the amino acid residues interacting with fructose at subsite - 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60mM GF2/GF3 by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117-127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF2. KEY POINTS: • Frischella β-fructofuranosidase hydrolyzed nystose more efficiently than 1-kestose. • Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference. • Loop consisting of residues 117-127 appears to contribute to the substrate binding.

Impacts of Apis cerana gut microbes on Nosema ceranae proliferation in Apis mellifera

The gut microbiota is a complex ecosystem including both beneficial and harmful microbes, which is essential for the metabolism, health and immunity of the host. Fecal microbiota transplantation (FMT) is an effective treatment for diseases of altered intestinal microbiota. Nosema ceranae, a parasitic fungus in intestinal epithelial cells, destroys the honey bee's gut integrity. This study provided the first report that the number of spores was significantly lower in the native host (Asian honey bee) compared with the novel host (European honey bee). The treatment effect of FMT was denoted by feeding the gut tissue solution from the original host to the new host. The results showed that FMT did not significantly decrease the spore load but enhanced the expression levels of immune genes in the Toll pathway. Our data confirmed that the native host inhibits N. ceranae proliferation. Our data suggest microbes of the native host could be an alternative approach to treat the parasite in the novel host.

Bioactive potential of invertase by yeast Saccharomyces cerevisiae from the honey bee gut: isolation and characterization

Bioactive potential of invertase by yeast Saccharomyces cerevisiae from the honey bee gut: isolation and characterization

Classic Hoarding Cages Increase Gut Bacterial Abundance and Reduce the Individual Immune Response of Honey Bee (Apis mellifera) Workers.

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.