Microbial ensemble in the hives: deciphering the intricate gut ecosystem of hive and forager bees of Apis mellifera.

BACKGROUND: The gut microbiome of honey bees significantly influences vital traits and metabolic processes, including digestion, detoxification, nutrient provision, development, and immunity. However, there is a limited information is available on the gut bacterial diversity of western honey bee populations in India. This study addresses the critical knowledge gap and outcome of which would benefit the beekeepers in India. METHODS AND RESULTS: This study investigates the gut bacterial diversity in forager and hive bees of Indian Apis mellifera, employing both culture-based and culture-independent methods. In the culturable study, a distinct difference in gut bacterial alpha and beta diversity between forager and hive bees emerges. Firmicutes, Proteobacteria, and Actinobacteria dominate, with hive bees exhibiting a Firmicutes-rich gut (65%), while foragers showcase a higher proportion of Proteobacteria (37%). Lactobacillus in the hive bee foregut aligns with the findings by other researchers. Bacterial amplicon sequencing analysisreveals a more intricate bacterial composition with 18 identified phyla, expanding our understanding compared to culturable methods. Hive bees exhibit higher community richness and diversity, likely due to diverse diets and increased social interactions. The core microbiota includes Snodgrassella alvi, Gilliamella apicola, and Bombilactobacillus mellis and Lactobacillus helsingborgensis, crucial for digestion, metabolism, and pathogen resistance. The study emphasises bacteria's role in pollen and nectar digestion, with specific groups like Lactobacillus and Bifidobobacterium spp. associated with carbohydrate metabolism and polysaccharide breakdown. These microbes aid in starch and sucrose digestion, releasing beneficial short-chain fatty acids. CONCLUSION: This research highlights the intricate relationship between honey bees and their gut microbiota, showcasing how the diverse and complex microbiome helps bees overcome dietary challenges and enhances overall host health. Understanding these interactions contributes to bee ecology knowledge and has implications for honey bee health management, emphasising the need for further exploration and conservation efforts.

Differences in the suitability of published honey bee (Apis mellifera) reference genes between the African subspecies Apis mellifera scutellata and European derived Apis mellifera.

Quantitative real-time polymerase chain reaction (qPCR) is a method widely used to determine changes and differences in gene expression. As target gene expression is most often quantified relative to the expression of reference genes, the validation of suitable reference genes is of critical importance. In practice, however, such validation might not be thoroughly conducted if the same species and the same tissue or body parts are used for qPCR experiments. Here we show, that qPCR reference genes published for workers of European honey bee (Apis mellifera) subspecies fail to be stably expressed in workers of the African subspecies Apis mellifera scutellata. This is the case even when the sampled workers are in the same life stage, the same organ was dissected and the same reagents were used. Thus, reference genes need to be thoroughly re-tested before they can be used as suitable references even when the only thing that changes is the subspecies used.

Assessing the Antimicrobial Properties of Honey Protein Components through In Silico Comparative Peptide Composition and Distribution Analysis.

The availability of reference proteomes for two honeybee species (Apis mellifera and Apis cerana cerana) opens the possibility of in silico studies of diverse properties of the selected protein fractions. The antimicrobial activity of honey is well established and related to its composition, including protein components. We have performed a comparative study on a selected fraction of the honey-related proteins, as well as other bee-secreted proteins, utilizing a publicly available database of established and verified peptides with antimicrobial properties. Using a high-performance sequence aligner (diamond), protein components with antimicrobial peptide sequences were identified and analyzed. The identified peptides were mapped on the available bee proteome sequences, as well as on model structures provided by the AlphaFold project. The results indicate a highly conserved localization of the identified sequences within a limited number of the protein components. Putative antimicrobial fragments also show high sequence-based similarity to the multiple peptides contained in the reference databases. For the 2 databases used, the lowest calculated percentage of similarity ranged from 30.1% to 32.9%, with a respective average of 88.5% and 79.3% for the Apis mellifera proteome. It was revealed that the antimicrobial peptides (AMPs) site is a single, well-defined domain with potentially conserved structural features. In the case of the examples studied in detail, the structural domain takes the form of the two ß-sheets, stabilized by α-helices in one case, and a six-ß-sheet-only domain localized in the C-terminal part of the sequence, respectively. Moreover, no significant differences were found in the composition of the antibacterial fraction of peptides that were identified in the proteomes of both species.

Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara.

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

Mechanism of the distinct toxicity level of imidacloprid and thiacloprid against honey bees: An in silico study based on cytochrome P450 9Q3.

The honey bee, Apis mellifera, shows variation in sensitivity to imidacloprid and thiacloprid, which does not reside at the target site but rather in the rapidly oxidative metabolism mediated by P450s (such as a single P450, CYP9Q3). An in silico study was conducted to investigate the various metabolism of imidacloprid and thiacloprid. The binding potency of thiacloprid was stronger and a stable π-π interaction with Phe121 and the N-H⋯N hydrogen bond with Asn214 are found in the CYP9Q3-thiacloprid system but absent in imidacloprid, which might affect the potential metabolic activity. Moreover, the values of highest occupied molecular orbit (HOMO) energy and the vertical ionization potential (IP) of two compounds demonstrated that thiacloprid is more likely to oxidation. The findings revealed the probable binding modes of imidacloprid and thiacloprid with CYP9Q3 and might facilitate future design of the low bee toxicity neonicotinoid insecticides.

Honeycomb, a New Food Resource with Health Care Functions: The Difference of Volatile Compounds found in Apis cerana and A. mellifera Honeycombs.

The honeycomb composition is very complex, containing honey, royal jelly, pollen, and propolis, and thus contains a large number of bioactive ingredients, such as polyphenols and flavonoids. In recent years, honeycomb as a new functional food resource has been favored by many bee product companies, but the basic research on honeycomb is lacking. The aim of this study is to reveal the chemical differences between A. cerana honeycombs (ACC) and A. mellifera honeycombs (AMC). In this paper, we studied the volatile organic components (VOCs) of ACC and AMC by solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS). A total of 114 VOCs were identified in 10 honeycombs. Furthermore, principal component analysis (PCA) revealed that the chemical composition of ACC and AMC were different. Additionally, orthogonal partial least squares discrimination analysis (OPLS-DA) revealed that benzaldehyde, octanal, limonene, ocimene, linalool, α-terpineol, and decanal are the significant VOCs in AMC extracts, which are mainly derived from propolis. OPLS-DA model also identified 2-phenylethanol, phenethyl acetate, isophorone, 4-oxoisophorone, betula, ethyl phenylacetate, ethyl palmitate, and dihydrooxophorone as potential discriminatory markers of ACC, which likely contribute to protecting the hive against microorganisms and keep it clean.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: Our 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. Video Abstract.

Honey bee populations of the USA display restrictions in their mtDNA haplotype diversity.

The genetic diversity of the USA honey bee (Apis mellifera L.) populations was examined through a molecular approach using two mitochondrial DNA (mtDNA) markers. A total of 1,063 samples were analyzed for the mtDNA intergenic region located between the cytochrome c oxidase I and II (COI-COII) and 401 samples were investigated for the NADH dehydrogenase 2 (ND2) coding gene. The samples represented 45 states, the District of Colombia and two territories of the USA. Nationwide, three maternal evolutionary lineages were identified: the North Mediterranean lineage C (93.79%), the West Mediterranean lineage M (3.2%) and the African lineage A (3.01%). A total of 27 haplotypes were identified, 13 of them (95.11%) were already reported and 14 others (4.87%) were found to be novel haplotypes exclusive to the USA. The number of haplotypes per state/territory ranged between two and eight and the haplotype diversity H ranged between 0.236-0.763, with a nationwide haplotype diversity of 0.597. Furthermore, the honey bee populations of the USA were shown to rely heavily (76.64%) on two single haplotypes (C1 = 38.76%, C2j = 37.62%) of the same lineage characterizing A. m. ligustica and A. m. carnica subspecies, respectively. Molecular-variance parsimony in COI-COII and ND2 confirmed this finding and underlined the central and ancestral position of C2d within the C lineage. Moreover, major haplotypes of A. m. mellifera (M3a, M7b, M7c) were recorded in six states (AL, AR, HI, MO, NM and WA). Four classic African haplotypes (A1e, A1v, A4, A4p) were also identified in nine states and Puerto Rico, with higher frequencies in southern states like LA, FL and TX. This data suggests the need to evaluate if a restricted mtDNA haplotype diversity in the US honey bee populations could have negative impacts on the beekeeping sustainability of this country.

Analytical Strategies for LC-MS-Based Untargeted and Targeted Metabolomics Approaches Reveal the Entomological Origins of Honey.

A comprehensive liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was developed to discriminate honey harvested from Apis mellifera ligustica Spinola (A. mellifera) and Apis cerana cerana Fabricius (A. cerana). Based on an untargeted strategy, ultrahigh-performance liquid chromatography electrospray ionization quadrupole orbitrap high-resolution mass spectrometry (UPLC Q-Orbitrap) was combined with chemometrics techniques to screen and identify tentative markers from A. mellifera and A. cerana honey. In targeted metabolomics analysis, a sensitive method of solid-phase extraction followed by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was established for quantifying three markers, and the results showed that 3-amino-2-naphthoic acid and methyl indole-3-acetate could be considered markers of A. cerana honey, as they were present in higher amounts in A. cerana honey than in A. mellifera honey, whereas kynurenic acid was determined to be a marker of A. mellifera honey. This work highlights critical information for the authentication of A. cerana and A. mellifera honey.

Genetic divergence and functional convergence of gut bacteria between the Eastern honey bee Apis cerana and the Western honey bee Apis mellifera.

Introduction: The functional relevance of intra-species diversity in natural microbial communities remains largely unexplored. The guts of two closely related honey bee species, Apis cerana and A. mellifera, are colonised by a similar set of core bacterial species composed of host-specific strains, thereby providing a good model for an intra-species diversity study. Objectives: We aim to assess the functional relevance of intra-species diversity of A. cerana and A. mellifera gut microbiota. Methods: Honey bee workers were collected from four regions of China. Their gut microbiomes were investigated by shotgun metagenomic sequencing, and the bacterial compositions were compared at the species level. A cross-species colonisation assay was conducted, with the gut metabolomes being characterised by LC-MS/MS. Results: Comparative analysis showed that the strain composition of the core bacterial species was host-specific. These core bacterial species presented distinctive functional profiles between the hosts. However, the overall functional profiles of the A. cerana and A. mellifera gut microbiomes were similar; this was further supported by the consistency of the honey bees' gut metabolome, as the gut microbiota of different honey bee species showed rather similar metabolic profiles in the cross-species colonisation assay. Moreover, this experiment also demonstrated that the gut microbiota of A. cerana and A. mellifera could cross colonise between the two honey bee species. Conclusion: Our findings revealed functional differences in most core gut bacteria between the guts of A. cerana and A. mellifera, which may be associated with their inter-species diversity. However, the functional profiles of the overall gut microbiomes between the two honey bee species converge, probably as a result of the overlapping ecological niches of the two species. Our findings provide critical insights into the evolution and functional roles of the mutualistic microbiota of honey bees and reveal that functional redundancy could stabilise the gene content diversity at the strain-level within the gut community.

The oral vaccination with Paenibacillus larvae bacterin can decrease susceptibility to American Foulbrood infection in honey bees-A safety and efficacy study.

Pollination services to increase crop production are becoming more and more important, as we are facing both climate change and a growing world population. Both are predicted to impact food security worldwide. High-density, commercial beekeeping has become a key link in the food supply chain, and diseases have become a central issue in hive losses around the world. American Foulbrood (AFB) disease is a highly contagious bacterial brood disease in honey bees (Apis mellifera), leading to hive losses worldwide. The causative agent is the Gram+ bacterium Paenibacillus larvae, which is able to infect honey bee larvae during the first 3 days of their lives. It can be found in hives around the world with viable spores for decades. Antibiotics are largely ineffective in treating the disease as they are only efficient against the vegetative state. Once a hive shows the clinical manifestation of the disease, the only effective way to eradicate it and prevent the spread of the disease is by burning the hive, the equipment, and the colony. Because of its virulent nature and detrimental effects on honey bee colonies, AFB is classified as a notifiable disease worldwide. Effective, safe, and sustainable methods are needed to ensure the wellbeing of honey bee colonies. Even though insects lack antibodies, which are the main requisites for trans-generational immune priming (TGIP), they can prime their offspring against persisting pathogens. Here, we demonstrate an increased survival of infected honey bee larvae after their queen was vaccinated, compared to offspring of control queens (placebo vaccinated). These results indicate that TGIP in insects can be used to majorly enhance colony health, protect commercial pollinators from deadly diseases, and reduce high financial and material losses to beekeepers. Classification: biological sciences, applied biological sciences.

Genetic diversity and phylogenetic relationship among the western and the Asian honey bees based on two mitochondrial gene segments (COI and ND5).

The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.

Foraging Behavior and Pollination Efficiency of Apis mellifera L. on the Oil Tree Peony 'Feng Dan' (Paeonia ostii T. Hong et J.X. Zhang).

To solve the issue of insufficient pollinating of insects for the oil tree peony 'Feng Dan' (Paeonia ostii T. Hong et J.X. Zhang) and improve its seed set and yield, we conducted observations from 2017 to 2018 to investigate the relationship between honey bee (Apis mellifera L.) foraging behavior and diurnal activity. We compared the single-fruit seed set ratio among three flower types on the same plants of the oil tree peony, which flowered simultaneously, in three pollination areas (bee pollination, natural field pollination, and controlled pollination by pollinators) and in a net room under self-pollination, wind pollination and bee pollination. Apis mellifera exhibited short single visitations, long visitations to a single flower and repeated visits to flowers of the oil tree peony. The number of flower visits of A. mellifera was significantly and positively yet weakly correlated with the number of stigma visits (2017: r = 0.045, p < 0.05; 2018: r = 0.195, p < 0.01). The seed set of oil tree peony follicles in the A. mellifera pollination area was significantly higher than that in the natural pollination field area and the control net rooms. On the same oil tree peony plant with synchronous flowering, the percent seed set of follicles pollinated by A. mellifera at a high density was significantly higher than that resulting from wind pollination and self-pollination.

Genetic and phylogenetic analysis of Chinese sacbrood virus isolates from Apis mellifera.

BACKGROUND: Sacbrood virus (SBV) is one of the most pathogenic honeybee viruses that exhibits host specificity and regional variations. The SBV strains that infect the Chinese honeybee Apis cerana are called Chinese SBVs (CSBVs). METHODS: In this study, a CSBV strain named AmCSBV-SDLY-2016 (GenBank accession No. MG733283) infecting A. mellifera was identified by electron microscopy, its protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and agar gel immunodiffusion assay, and its nucleotide sequence was identified using a series of reverse-transcription polymerase chain reaction fragments of AmCSBV-SDLY-2016 generated using SBV/CSBV-specific primers. To investigate phylogenetic relationships of the CSBV isolates, a phylogenetic tree of the complete open reading frames (ORF) of the CSBV sequences was constructed using MEGA 6.0; then, the similarity and recombination events among the isolated CSBV strains were analyzed using SimPlot and RDP4 software, respectively. RESULTS: Sequencing results revealed the complete 8,794-nucleotide long complete genomic RNA of the strain, with a single large ORF (189-8,717) encoding 2,843 amino acids. Comparison of the deduced amino acid sequence with the SBV/CSBV reference sequences deposited in the GenBank database identified helicase, protease, and RNA-dependent RNA polymerase domains; the structural genes were located at the 5' end, whereas the non-structural genes were found at the 3' end. Multiple sequence alignment showed that AmCSBV-SDLY-2016 had a 17-amino acid (aa) and a single aa deletion at positions 711-729 and 2,128, respectively, as compared with CSBV-GD-2002, and a 16-aa deletion (positions 711-713 and 715-728) as compared with AmSBV-UK-2000. However, AmCSBV-SDLY-2016 was similar to the CSBV-JLCBS-2014 strain, which infects A. cerana. AmCSBV-SDLY-2016 ORF shared 92.4-97.1% identity with the genomes of other CSBV strains (94.5-97.7% identity for deduced amino acids). AmCSBV-SDLY-2016 was least similar (89.5-90.4% identity) to other SBVs but showed maximum similarity with the previously reported CSBV-FZ-2014 strain. The phylogenetic tree constructed from AmCSBV-SDLY-2016 and 43 previously reported SBV/CSBV sequences indicated that SBV/CSBV strains clustered according to the host species and country of origin; AmCSBV-SDLY-2016 clustered with other previously reported Chinese and Asian strains (AC genotype SBV, as these strains originated from A. cerana) but was separate from the SBV genomes originating from Europe (AM genotype SBV, originating from A. mellifera). A SimPlot graph of SBV genomes confirmed the high variability, especially between the AC genotype SBV and AM genotype SBV. This genomic diversity may reflect the adaptation of SBV to specific hosts, ability of CSBV to cross the species barrier, and the spatial distances that separate CSBVs from other SBVs.

FoxP in bees: A comparative study on the developmental and adult expression pattern in three bee species considering isoforms and circuitry.

Mutations in the transcription factors FOXP1, FOXP2, and FOXP4 affect human cognition, including language. The FoxP gene locus is evolutionarily ancient and highly conserved in its DNA-binding domain. In Drosophila melanogaster FoxP has been implicated in courtship behavior, decision making, and specific types of motor-learning. Because honeybees (Apis mellifera, Am) excel at navigation and symbolic dance communication, they are a particularly suitable insect species to investigate a potential link between neural FoxP expression and cognition. We characterized two AmFoxP isoforms and mapped their expression in the brain during development and in adult foragers. Using a custom-made antiserum and in situ hybridization, we describe 11 AmFoxP expressing neuron populations. FoxP was expressed in equivalent patterns in two other representatives of Apidae; a closely related dwarf bee and a bumblebee species. Neural tracing revealed that the largest FoxP expressing neuron cluster in honeybees projects into a posterior tract that connects the optic lobe to the posterior lateral protocerebrum, predicting a function in visual processing. Our data provide an entry point for future experiments assessing the function of FoxP in eusocial Hymenoptera.

Chinese sacbrood virus infection in Apis mellifera, Shandong, China, 2016.

The Chinese sacbrood virus (CSBV) was first isolated from Apis cerana in 1972. However, the biological characteristics of the CSBV that naturally infects Apis mellifera, causing larval death, have not been reported yet. In the present study, natural CSBV infection was evaluated using clinical symptoms of A. mellifera larvae, RT-PCR, electron microscopy, agar gel immunodiffusion assays, and virus analysis in inoculated A. cerana larvae. The isolated CSBV strain was named AmCSBV-SDLY-2016. Subsequently, AmCSBV-SDLY-2016 was analyzed by constructing a phylogenetic tree using VP1. Data from the phylogenetic tree suggested that AmCSBV-SDLY-2016 is evolutionarily close to JLCBS-2014. It was also observed that CSBV crossed the species barrier, causing the death of A. mellifera larvae.