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.

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.

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.

Lipidomic Profiling Reveals Distinct Differences in Sphingolipids Metabolic Pathway between Healthy Apis cerana cerana larvae and Chinese Sacbrood Disease.

Chinese sacbrood disease (CSD), which is caused by Chinese sacbrood virus (CSBV), is a major viral disease in Apis cerana cerana larvae. Analysis of lipid composition is critical to the study of CSBV replication. The host lipidome profiling during CSBV infection has not been conducted. This paper identified the lipidome of the CSBV-larvae interaction through high-resolution mass spectrometry. A total of 2164 lipids were detected and divided into 20 categories. Comparison of lipidome between healthy and CSBV infected-larvae showed that 266 lipid species were altered by CSBV infection. Furthermore, qRT-PCR showed that various sphingolipid enzymes and the contents of sphingolipids in the larvae were increased, indicating that sphingolipids may be important for CSBV infection. Importantly, Cer (d14:1 + hO/21:0 + O), DG (41:0e), PE (18:0e/18:3), SM (d20:0/19:1), SM (d37:1), TG (16:0/18:1/18:3), TG (18:1/20:4/21:0) and TG (43:7) were significantly altered in both CSBV_24 h vs. CK_24 h and CSBV_48 h vs. CK_48 h. Moreover, TG (39:6), which was increased by more than 10-fold, could be used as a biomarker for the early detection of CSD. This study provides evidence that global lipidome homeostasis in A. c. cerana larvae is remodeled after CSBV infection. Detailed studies in the future may improve the understanding of the relationship between the sphingolipid pathway and CSBV replication.

Differential Viral Distribution Patterns in Reproductive Tissues of Apis mellifera and Apis cerana Drones.

Honeybee drones are male bees that mate with virgin queens during the mating flight, consequently transferring their genes to offspring. Therefore, the health of drones affects the overall fitness of the offspring and ultimately the survivability of the colony. Honeybee viruses are considered to be a major threat to the health of honeybees. In the present study, we demonstrated the pattern of common honeybee viruses in various tissues of drones in the western honeybee, Apis mellifera, and the eastern honeybee, Apis cerana. Drones were collected during the mating flight and analyzed using quantitative real-time (qRT-PCR) to detect the presence of seven honeybee viruses. The qRT-PCR result revealed that three honeybee viruses, namely Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Chinese Sacbrood Virus (CSBV), were detected in the reproductive tissues of A. mellifera and A. cerana drones. The results from qRT-PCR showed that the Israeli Acute Paralysis Virus (IAPV) was only detected in A. mellifera drone body tissues. Moreover, the prevalence of DWV and BQCV in the drones collected from A. mellifera colonies was significantly higher than that of A. cerana. In addition, virus multiple infections were higher in A. mellifera drones compared to those in A. cerana. CSBV was found predominantly in the reproductive tissues of A. cerana drones. This study is the first report describing the presence of the CSBV in reproductive tissues of A. mellifera drones. Our results may reflect the preference of honeybee viruses in honeybee species and may provide a piece of interesting evidence for understanding the virus transmission in A. cerana.

The role of melatonin and Tryptophan-5-hydroxylase-1 in different abiotic stressors in Apis cerana cerana.

Tryptophan-5-hydroxylase-1 (T5H-1) is the rate-limiting enzyme in the biosynthesis of serotonin, which is involved in the biosynthesis of melatonin (Mel). Mel, a biological hormone, plays crucial roles in stressors tolerance, such as cold, hot, Ultraviolet (UV) and pesticide tolerance. However, the direct correlation between T5H-1 and Mel and the underlying mechanism in organisms remains elusive. Mel-mediated cold tolerance was studied extensively in plants and somewhat in insects, including bees. The present study isolated the Mel synthesis gene T5H-1 from Apis cerana cerana for the first time. qRT-PCR analysis indicated that AccT5H-1 played vital roles during some adverse conditions, including 4 °C, 8 °C, 10 °C, 45 °C, UV, cyhalothrin, abamectin, paraquat and bifenthrin exposure. Knockdown of AccT5H-1 using RNA interference (RNAi) technology upregulated most antioxidant genes. Additionally, an enzyme activity assay revealed higher contents of Malondialdehyde (MDA) and Hydrogen peroxide (H2O2), lower content of Vitamin C (VC), and higher activities of Glutathione S-transferase (GST), Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) in the AccT5H-1 silenced group than the control group. These results suggest that AccT5H-1 is involved in the response to different oxidative stressors in A. cerana cerana. The survival rate of A. cerana cerana exposed to low temperature treatment revealed that the optimal concentration of Mel in the diet was 10 µg/mL. We also found that the antioxidant enzyme (GST, SOD, POD and CAT) concentrations at 10 µg/mL Mel increased to different degrees, and the content of oxidizing substances (MDA and H2O2) decreased, the content of VC increased, and the content of substances that promote cold resistance (glycerol and glycogen) increased. Mel increased the resistance of A. cerana cerana exposed to low temperatures. The expression of AccT5H-1 decreased after the feeding of exogenous Mel to bees. These results provide a reference for other insect studies on Mel and T5H-1.

Multienzyme and Antibacterial Potential of Bacteria Isolated from gut of Asian Honey Bee (Apis cerana Indica), Lahore Using Culture Dependent Method

Abstract The bacteria residing in the gut of honey bees (HB) has demonstrated a significant role in protecting bees against various pathogens, production of honey and wax. However, no information exists about the antibacterial potential of bacterial isolates from gut of Asian HB, Apis cerana Indica F. (Hymenoptera: Apidae), against human pathogens. This study aims to investigate the antibacterial and multienzyme potential of aerobic bacteria from A. cerana gut using culture dependent approach. A total of 12 HB gut bacteria were characterized morphologically and biochemically. These strains were further screened for their antimicrobial activity against pathogenic human microorganisms Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus licheniformis and Bacillus subtilis using cross streak (primary screening) and agar well diffusion methods (secondary screening). Preliminary characterization of cell-free supernatant (CFS) of two promising isolates was performed by measuring lactic acid concentrations, enzymatic digestion of antimicrobial compounds, stability over a range of temperature, pH and amplification of spaS (subtilin) and spoA (subtilosin) genes. In primary screening, among 12 HB isolates, eight strains showed statistically significant highest zones of inhibition (p≤0.05) against E. coli, K. pneumoniae and P. aeruginosa. 16S rRNA sequencing revealed that these isolates belong to Bacillus genus, identified as B. tequilensis, B. pumilus, B. xiamenensis, B. subtilis, B. amyloliquefaciens, B. safensis, B. licheniformis, B. altitudinis (Accession numbers: MT186230-MT186237). Secondary screening revealed that among eight isolates, B. subtilis and B. amyloliquefaciens showed statistically significantly strong inhibition (p≤0.05) against all tested pathogens. Antibiotic susceptibility testing revealed that both isolates were resistant to antibiotics and possesses proteolytic, lipolytic and cellulolytic activities. The nature of the compound causing inhibitory activity was found to be proteinaceous and showed stability over a wide range of temperature as well as pH. PCR study confirmed the presence of bacteriocins by successful amplification of important antimicrobial peptide biosynthesis genes spaS and spoA. These results suggest that the HB gut is a home to bacteria that possess antimicrobial activity and important enzymes with antimicrobial potential. To our knowledge, this is the first report demonstrating the antimicrobial potential of bacteria isolated from gut of HB (A. cerana) against human pathogens.

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.

Hepatoprotective Effects of the Honey of Apis cerana Fabricius on Bromobenzene-Induced Liver Damage in Mice.

Apis cerana honey (honey of Apis cerana Fabricius), widely distributed in the mountain areas of East Asia, has not been studied fully. The hepatoprotective activity of A. cerana honey was evaluated against bromobenzene-induced liver damage in mice. In high dose, A. cerana honey can significantly alleviate liver injury, as is indicated by the depressed levels of serum alanine aminotransferase (ALT) (59.13%) and aspartate aminotransferase (AST) (79.71%), the inhibited malondialdehyde (MDA) content (63.30%), the elevated activities of superoxide dismutase (SOD) (73.12%) and glutathione-Px (57.24%), and the decreased expression of Transforming growth factor ß1 (51.83%) induced by bromobenzene (P < 0.05). The quantitative analysis of twelve major constituents (1 to 12) of A. cerana honey was executed by high performance liquid chromatography-diode array detector. The results indicate that treatment with A. cerana honey can prevent bromobenzene-induced hepatic damage in mice. Polyphenols might be the bioactive substances attributed to its antioxidant properties and intervention of oxidative stress.

Antioxidant and hepatoprotective effects of A. cerana honey against acute alcohol-induced liver damage in mice.

A. cerana honey, gathered from Apis cerana Fabricius (A. cerana), has not been fully studied. Samples of honey originating from six geographical regions (mainly in the Qinling Mountains of China) were investigated to determine their antioxidant and hepatoprotective effects against acute alcohol-induced liver damage. The results showed that A. cerana honeys from the Qinling Mountains had high total phenolic contents (345.1-502.1mgGAkg-1), ascorbic acid contents (153.8-368.4mgkg-1), and strong antioxidant activities in DPPH radical scavenging activity assays (87.5-136.2IC50mgmL-1), ferric reducing antioxidant powers (191.8-317.4mgTroloxkg-1), and ferrous ion-chelating activities (27.5-35.5mgNa2EDTAkg-1). Pretreatment with A. cerana honey (Qinling Mountains) at 5, 10, or 20gkg-1 twice daily for 12weeks significantly inhibited serum lipoprotein oxidation and increased serum radical absorbance capacity (ORAC) (P<0.05). Moreover, A. cerana honey inhibited acute alcohol-induced increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum (P<0.05), reduced the production of hepatic malondialdehyde (MDA) (P<0.05), and promoted superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities (P<0.05). More importantly, it also remarkably inhibited the level of TGF-ß1 in the serum and liver (P<0.05). The results of this study indicate that administration of A. cerana honey prevents acute alcohol-induced liver damage likely because of its antioxidant properties and ability to prevent oxidative stress.

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.