The eastern (Apis cerana cerana, Acc) and western (Apis mellifera ligustica, Aml) honeybee are two major honeybee species. Surprisingly, little is known about the fundamental molecular neurobiology of brain suborgans of Acc and Aml. We characterized and compared the proteomes of mushroom bodies (MBs), antennal lobes (ALs) and optical lobes (OLs) in the brain of both species, and biologically validated the functions related to learning and memory. Acc and Aml have evolved similar proteome signatures in MBs and OLs to drive the domain-specific neural activities. In MBs of both species, commonly enriched and enhanced functional groups related to protein metabolism and Ca²⁺ transport relative to ALs and OLs, suggests that proteins and Ca²⁺ are vital for consolidating learning and memory via modulation of synaptic structure and signal transduction. Furthermore, in OLs of both species, the mainly enriched ribonucleoside metabolism suggests its vital role as second messenger in promoting phototransduction. Notably, in ALs of both species, distinct proteome settings have shaped to prime olfactory learning and memory. In ALs of Acc, this is supported by the enriched cytoskeleton organization to sustain olfactory signaling through modulation of plasticity in glomeruli and intracellular transport. In ALs of Aml, however, the enriched functional groups implicated in hydrogen ion transport are indicative of their importance in supporting olfactory processes by regulation of synaptic transmission. The biological confirmation of enhanced activities of protein metabolism and signal transduction in ALs and MBs of Acc relative to in Aml demonstrates that a stronger sense of olfactory learning and memory has evolved in Acc. The reported first in-depth proteome data of honeybee brain suborgans provide a novel insight into the molecular basis of neurobiology, and is potentially useful for further neurological studies in honeybees and other insects.

Bee pollen as a nutrient-rich functional food has been considered for use as an adjuvant for chronic disease therapy. However, bee pollen can trigger food-borne allergies, causing a great concern to food safety. Our previous study demonstrated that the combined use of cellulase, pectinase and papain can hydrolyze allergens into peptides and amino acids, resulting in reduced allergenicity of bee pollen based on in vitro assays. Herein, we aimed to further explore the mechanisms behind allergenicity alleviation of enzyme-treated bee pollen through a BALB/c mouse model. Results showed that the enzyme-treated bee pollen could mitigate mice scratching frequency, ameliorate histopathological injury, decrease serum IgE level, and regulate bioamine production. Moreover, enzyme-treated bee pollen can modulate metabolic pathways and gut microbiota composition in mice, further supporting the alleviatory allergenicity of enzyme-treated bee pollen. The findings could provide a foundation for further development and utilization of hypoallergenic bee pollen products.

Royal jelly (RJ), a natural honeybee product, has a wide range of antibacterial activities. N-glycosylated major royal jelly protein 2 (N-MRJP2), purified from RJ, can inhibit the growth of Paenibacillus larvae (P. larvae, Gram-positive), a contagious etiological agent of the American foulbrood disease of honeybees. However, the inhibitory mechanism is largely unknown. Antibacterial assay and membrane proteome were conducted to investigate the inhibition capacity of RJ from different instar larvae and P. larvae treated by N-MRJP2, respectively. The similar antibacterial efficiency of RJ from different larval instar indicates that RJ is vital for the adaptive immune defense of small larvae. The killing of P. larvae by N-MRJP2 is achieved by disturbing the cell wall biosynthesis, increasing the permeability of cell membrane, hindering aerobic respiration, restraining cell division and inducing cell death. This demonstrates that RJ is critical for the passive immunity of immature larvae and N-MRJP2 can be used as natural antibiotic substance to resist P. larvae, even for other gram-positive bacteria. This constitutes solid evidence that RJ and N-MRJP2 have potentials as novel antibacterial agents.

Ascosphaera apis is the causative agent of honey bee chalkbrood disease, and spores are the only known source of infections. Interference with sporulation is therefore a promising way to manage A. apis. The versicolorin reductase gene (StcU-2) is a ketoreductase protein related to sporulation and melanin biosynthesis. To study the StcU-2 gene in ascospore production of A. apis, CRISPR/Cas9 was used, and eight hygromycin B antibiotic-resistant transformants incorporating enhanced green fluorescent protein (EGFP) were made and analyzed. PCR amplification, gel electrophoresis, and sequence analysis were used for target gene editing analysis and verification. The CRISPR/Cas9 editing successfully knocked out the StcU-2 gene in A. apis. StcU-2 mutants had shown albino and non-functional spore-cyst development and lost effective sporulation. In conclusion, editing of StcU-2 gene has shown direct relation with sporulation and melanin biosynthesis of A. apis; this effective sporulation reduction would reduce the spread and pathogenicity of A. apis to managed honey bee. To the best of our knowledge, this is the first time CRISPR/Cas9-mediated gene editing has been efficiently performed in A. apis, a fungal honey bee brood pathogen, which offers a comprehensive set of procedural references that contributes to A. apis gene function studies and consequent control of chalkbrood disease.