Defining honeybee subspecies in an evolutionary context warrants strategized conservation.

Despite the urgent need for conservation consideration, strategic action plans for the preservation of the Asian honeybee, Apis cerana Fabricius, 1793, remain lacking. Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy. Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts, as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities. Here, we investigated genome variations in 362 worker bees representing almost all populations of mainland A. cerana to understand how evolution has shaped its population structure. Whole-genome single nucleotide polymorphisms (SNPs) based on nuclear sequences revealed eight putative subspecies, with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies. Our results demonstrated that most classic morphological traits, including body size, were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism. Thus, such morphological traits were not suitable for subspecific delineation. Conversely, wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes. Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor. Based on our findings, we propose that criteria for subspecies delineation should be based on evolutionary independence, trait distinction, and geographic isolation. We formally defined and described eight subspecies of mainland A. cerana. Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units, guiding colony introduction and breeding.

Significant compositional and functional variation reveals the patterns of gut microbiota evolution among the widespread Asian honeybee populations.

The gut microbiome is a crucial element that facilitates a host's adaptation to a changing environment. Compared to the western honeybee Apis mellifera, the Asian honeybee, Apis cerana populations across its natural range remain mostly semi-feral and are less affected by bee management, which provides a good system to investigate how gut microbiota evolve under environmental heterogeneity on large geographic scales. We compared and analyzed the gut microbiomes of 99 Asian honeybees, from genetically diverged populations covering 13 provinces across China. Bacterial composition varied significantly across populations at phylotype, sequence-discrete population (SDP), and strain levels, but with extensive overlaps, indicating that the diversity of microbial community among A. cerana populations is driven by nestedness. Pollen diets were significantly correlated with both the composition and function of the gut microbiome. Core bacteria, Gilliamella and Lactobacillus Firm-5, showed antagonistic turnovers and contributed to the enrichment in carbohydrate transport and metabolism. By feeding and inoculation bioassays, we confirmed that the variations in pollen polysaccharide composition contributed to the trade-off of these core bacteria. Progressive change, i.e., nestedness, is the foundation of gut microbiome evolution among the Asian honeybee. Such a transition during the co-diversification of gut microbiomes is affected by environmental factors, diets in general, and pollen polysaccharides in particular.

Community Dynamics in Structure and Function of Honey Bee Gut Bacteria in Response to Winter Dietary Shift.

Temperate honey bees (Apis mellifera) are challenged by low temperatures and abrupt dietary shifts associated with behavioral changes during winter. Case studies have revealed drastic turnover in the gut microbiota of winter bees, highlighted by the seasonal dominance of a non-core bacterium Bartonella. However, neither biological consequence nor underlying mechanism of this microbial turnover is clear. In particular, we ask whether such changes in gut profile are related to winter dietary shift and possibly beneficial to host and associated gut microbiome? Here, we integrated evidences from genomics, metagenomics, and metabolomics in three honey bee subspecies maintained at the same locality of northern China to profile both diversity and functional variations in gut bacteria across seasons. Our results showed that winter dominance of Bartonella was shared in all tested honey bee lineages. This seasonal change was likely a consequence of winter dietary shifts characterized by greatly reduced pollen consumption and accumulation of metabolic waste due to restricted excretion. Bartonella showed expanded genomic capacity in utilizing more diverse energy substrates, such as converting metabolic wastes lactate and ethanol into pyruvate, an energy source for self-utilization and possibly also for host and other symbionts. Furthermore, Bartonella was the only bacterium capable of both producing and secreting tryptophan and phenylalanine, whose metabolic products were detected in bee guts, even though all gut bacteria lacked relevant digestion enzymes. These results thus suggested a possible mechanism where the gut bacteria might benefit the host by supplementing them with essential amino acids lacking in a protein shortage diet. IMPORTANCE The abilities to survive winter and to adapt to major food changes are key traits that have enabled successful range expansion of the honey bees from the tropic to temperate climate. Our results highlighted a new possibility that gut bacteria may have played an important role in host survival of the severe winter condition. The non-core bacterium Bartonella is not only more adaptive to the winter diet but is also equipped with the capacity to provide the host with essential nutrients and important metabolic substrates. This overall host-bacterium profile is probably favored by natural selection, resulting in a consistent winter gut strategy across varied honey bee lineages. Conversely, when the hosts start to forage again, core bacteria maintained at low abundance during winter returned to their typical dominant status, thus completing the annual gut turnover. Our study suggests a new hypothesis where seasonal gut variations may improve the fitness of the honey bee, allowing them to explore more diverse climates.

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.

Full length transcriptomes analysis of cold-resistance of Apis cerana in Changbai Mountain during overwintering period.

Apis cerana in Changbai Mountain is an ecological type of Apis cerana, which is an excellent breeding material with cold-resistant developed by long-term natural selection under the ecological conditions. However, the physiological and molecular mechanisms of Changbai Mountain population under cold stress are still unclear. In this study, the Nanopore sequencing was carried out for the transcriptome of Apis cerana in Changbai Mountain in the coldest period of overwintering, which will provide a reference to the cold-resistant mechanism. We determined 5,941 complete ORF sequences, 1,193 lncRNAs, 619 TFs, 10,866 SSRs and functional annotations of 11,599 new transcripts. Our results showed that the myosin family and the C2H2 zinc finger protein transcription factor family possibly have significant impacts on the response mechanism of cold stress during overwintering. In addition, the cold environment alters genes expression profiles in honeybees via different AS and APA mechanisms. These altered genes in Hippo, Foxo, and MARK pathways help them counter the stress of cold in overwinter period. Our results might provide clues about the response of eastern honeybees to extreme cold, and reflect the possible genetic basis of physiological changes.

Free fatty acids induce the demethylation of the fructose 1,6-biphosphatase 2 gene promoter and potentiate its expression in hepatocytes.

Obesity is a serious health issue as it is a social burden and the main risk factor for other metabolic diseases. Increasing evidence indicates that a high-fat diet (HFD) is the key factor for the development of obesity, but the key genes and their associated molecular mechanisms are still not fully understood. In this study, we performed integrated bioinformatic analysis and identified that fructose-1,6 biphosphatase 2 (FBP2) was involved in free fatty acids (FFAs)-induced lipid droplet accumulation in hepatocytes and HFD-induced obesity in mice. Our data showed that palmitate (PA) and oleic acid (OA) induced the expression of FBP2 in time- and dose-dependent manners, and accelerated the development of lipid droplets in LO2 human normal liver cells. In HFD-fed C57BL/6 mice, accompanied by insulin resistance and lipid droplet accumulation, the mRNA and protein levels of FBP2 in the livers also increased significantly. The results from the methylation sequencing PCR (MSP) and bisulfite specific PCR (BSP) indicated that PA/OA induced the demethylation of the FBP2 gene promoter in LO2 cells. Moreover, betaine, a methyl donor, attenuated the expression of the FBP2 gene, the accumulation of lipid droplets, and the expression of perilipin-2, a biomarker of lipid droplets, in LO2 cells. All these findings revealed that FBP2 might be involved in HFD-induced obesity, and it is of interest to investigate the role of FBP2 in the treatment and prevention of obesity and its associated complications.

Gene reuse facilitates rapid radiation and independent adaptation to diverse habitats in the Asian honeybee.

Animals with recent shared ancestry frequently adapt in parallel to new but similar habitats, a process often underlined by repeated selection of the same genes. Yet, in contrast, few examples have demonstrated the significance of gene reuse in colonization of multiple disparate habitats. By analyzing 343 genomes of the widespread Asian honeybee, Apis cerana, we showed that multiple peripheral subspecies radiated from a central ancestral population and adapted independently to diverse habitats. We found strong evidence of gene reuse in the Leucokinin receptor (Lkr), which was repeatedly selected in almost all peripheral subspecies. Differential expression and RNA interference knockdown revealed the role of Lkr in influencing foraging labor division, suggesting that Lkr facilitates collective tendency for pollen/nectar collection as an adaptation to floral changes. Our results suggest that honeybees may accommodate diverse floral shifts during rapid radiation through fine-tuning individual foraging tendency, a seemingly complex process accomplished by gene reuse.

Trilobatin, a Novel SGLT1/2 Inhibitor, Selectively Induces the Proliferation of Human Hepatoblastoma Cells.

Studies have indicated that Na+-d-glucose co-transporter (SGLT) inhibitors had anti-proliferative activity by attenuating the uptake of glucose in several tumor cell lines. In this study, the molecular docking showed that, trilobatin, one of the dihydrochalcones from leaves of Lithocarpus polystachyus Rehd., might be a novel inhibitor of SGLT1 and SGLT2, which evidently attenuated the uptake of glucose in vitro and in vivo. To our surprise, we observed that trilobatin did not inhibit, but promoted the proliferation of human hepatoblastoma HepG2 and Huh 7 cells when it was present at high concentrations. At the same time, incubation with high concentrations of trilobatin arrested the cell cycle at S phase in HepG2 cells. We also found that treatment with trilobatin had no significant effect on the expression of hepatitis B x-interacting protein (HBXIP) and hepatocyte nuclear factor (HNF)-4α, the two key regulators of hepatocyte proliferation. Taken together, although trilobatin worked as a novel inhibitor of SGLTs to attenuate the uptake of glucose, it also selectively induced the cell proliferation of HepG2 cells, suggesting that not all the SGLT inhibitors inhibited the proliferation of tumor cells, and further studies are needed to assess the anti-cancer potentials of new glucose-lowering agents.

Contribution of lipids in honeybee (Apis mellifera) royal jelly to health.

Honeybee (Apis mellifera) royal jelly (RJ) has a long history in human medicine because of its health-protecting properties. To develop a fundamental and comprehensive understanding of lipids in RJ, this article reviews the available literature on lipid compounds identified from RJ extracts and in vitro pharmacological effects of 10-hydroxy-2-decenoic acid in RJ and other closely related compounds, some of which are also identified as lipid compounds in RJ. Overall, the lipids in RJ are composed of mostly (aliphatic) fatty acids, almost all of which are present as free fatty acids and scarcely any as esters. Most fatty acids in RJ are medium-chain fatty acids, whether hydroxylated in terminal and/or internal positions, terminated with mono- or dicarboxylic acid groups, and saturated or monounsaturated at the 2-position. Besides these fatty acids, lipids in RJ contain sterols in minor amounts. Lipids in RJ are useful as preventive and supportive medicines with functionalities that include potential inhibitors of cancer growth, immune system modulators, alternative therapies for menopause, skin-aging protectors, neurogenesis inducers, and more. Taken together, the evidence suggests that health-protecting properties of RJ can be, in part, ascribed to actions of lipids in RJ.

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI.

BACKGROUND: Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. RESULTS: To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. CONCLUSIONS: It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis.

Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled. Proteomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 microM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and calcium-binding protein 1 were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.