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Abstract: Little attention has been paid to the drone honeybee (Apis mellifera ligustica) which is a haploid individual carrying only the set of alleles that it inherits from its mother. Molecular mechanisms underlying drone embryogenesis are poorly understood. This study evaluated protein expression profiles of drone embryogenesis at embryonic ages of 24, 48 and 72h. More than 100 reproducible proteins were analyzed by mass spectrometry on 2D electrophoresis gels. Sixty-two proteins were significantly changed at the selected three experimental age points. Expression of the metabolic energy requirement-related protein peaked at the embryonic age of 48h, whereas development and metabolizing amino acid-related proteins expressed optimally at 72h. Cytoskeleton, protein folding and antioxidant-related proteins were highly expressed at 48 and 72h. Protein networks of the identified proteins were constructed and protein expressions were validated at the transcription level. This first proteomic study of drone embryogenesis in the honeybee may provide geneticists an exact timetable and candidate protein outline for further manipulations of drone stem cells. [Copyright &y& Elsevier]

Age-based division of labor among workers is a fundamental life-history trait of many social insects, including the Western honey bee, Apis mellifera L. Extensive studies of the causation of the most pronounced transition from performing tasks in the nest to outside foraging indicate hormonal regulation of complex physiological changes. However, the proximate neurobiological mechanisms that cause the behavioral repertoire to change are still not understood and require novel approaches to be fully characterized. Thus, we established the first comprehensive monoclonal antibody microarray in honey bees with 16,320 antibodies to directly identify proteins in the brain that regulate the transition to foraging. Major royal jelly protein (MRJP) 1 and MRJP3 were identified as potential protein effectors and further investigated. A series of experimental manipulations of the workers' behavioral transition led to changes in MRJP1 and MRJP3 quantities in accordance with their presumed functional role. Injection of MRJPs into the brain resulted in increased task-reversal from foraging to nursing and decreased task-progression from nursing to foraging, while the latter was increased by injection with MRJP antibodies. Finally, down-regulation of MRJP1 and MRJP3 expression via RNAi injection into the brain increased the transition from in-hive nursing to outside foraging, confirming a causal role of these two proteins in the proximate regulation of behavior and life-history of honey bee workers. Interaction partners of MRJP1 and MRJP3 in the honey bee brain included other regulators of honey bee behavior and life history. Thus, our transformative methodological advancement of proteome analysis in honey bees reveals novel regulators of honey bee behavior, extends our understanding of the functional pleiotropy of MRJPs, and supports a general nutrition-based model of the regulation of the age-based division of labor in honey bees. [Display omitted] [ABSTRACT FROM AUTHOR]

The honeybee species A. mellifera and A. cerana have evolved substantial differences in olfactory-driven behaviors and in peripheral olfactory systems. Knowledge of the central nervous system regulating these olfaction differences is limited, however. We compared the phosphoproteome of the antennal lobes (ALs, the primary olfactory neuropil) of A. mellifera and A. cerana , and identified a total of 2812 phosphopeptides carrying 2971 phosphosites from 1265 phosphoproteins. Of these, 76% of the phosphoproteins were shared by both species, which were mainly presynapse and cytoskeleton components, and were involved in signal transduction and neurotransmitter secretion. This finding indicates the fundamental role of protein phosphorylation in regulating signal transduction in the ALs. The mTOR signaling pathway, the phagosome pathway, and the autophagy pathway, which are important in protein metabolism, were enriched, suggesting glomeruli plasticity and olfactory processing are intensively modulated by phosphorylation via these pathways. Compared with A. mellifera , 107 phosphoproteins associated with protein metabolism and transport were uniquely expressed in A. cerana , indicating the protein synthesis-dependent synaptic plasticity is enhanced in A. cerana to facilitate the processing of more complex floral odor clues in mountain foraging areas. This finding is further supported by the significantly upregulated key phosphoproteins of the mTOR signaling pathway in A. cerana ALs. These results provide insights into the phosphoproteomic basis of neuroplasticity that is coupled with the divergent evolution of bees in different environments. To adapt to their own ecological niche, the two major honeybee species, A. mellifera and A. cerana , have developed significant difference in olfactory-driven behaviors, but our understanding of the underlying regulation of the central nervous system is still limitate. Here we performed the first comprehensive phosphoproteomic comparison of antennal lobes (Als) between A. mellifera and A. cerena. A large proportion of the identified phosphosites and phosphoproteins were shared between the two species to serve as a core network in the regulation of signal transduction and glomeruli plasticity of ALs. However, compared with A. mellifera , 107 phosphoproteins associated with protein metabolism and transport were uniquely identified in A. cerana ALs, and also several key phosphoproteins in mTOR signaling pathway were found upregulated in A. cerana. These findings indicate protein phosphorylation enhanced the protein synthesis-dependent synaptic plasticity in A. cerana to facilitate the processing of more complex floral odor clues in mountain foraging areas. Our data provide a valuable insight into phosphoproteome-driven cerebral regulation of honeybee olfactory behaviors, which is potentially useful for further neurobiological investigation in both honeybees and other insects. [Display omitted] • Phosphoproteins of honeybee antenal lobes are mainly functioning in neurotransmitter transport and signal transduction. • Protein phosphorylation significantly mediates the ion channel activity and glomeruli plasticity in antenal lobes • Protein phosphorylation is conserved in regulating olfactory process of both A. mellifera and A. cerena • Phosphorylation in the mTOR signaling pathway plays a vital role in olfactory divergence between A. mellifera and A. cerena. [ABSTRACT FROM AUTHOR]

Abstract: Honeybee salivary glands (SGs) are important exocrine glands. However, the molecular basis of how SGs fulfill their biological duty is still elusive. Proteomics and phosphoproteomics of cephalic SG (HSG) and thoracic SG (TSG) were compared between normal and single-cohort honeybee colonies. Of 113 and 64 differentially regulated proteins and phosphoproteins, 86 and 33 were identified, respectively. The SGs require a wide spectrum of proteins to support their multifaceted functions and ensure normal social management of the colony. Changes of protein expression and phosphoproteins are key role players. The HSG triggers labor transition from in-hive work to foraging activities via the regulation of juvenile hormone and ethyl oleate levels. The stronger expression of proteins involved in carbohydrate and energy metabolism, protein folding, protein metabolism, cellular homeostasis and cytoskeleton in TSG, supports the gland to efficiently enhance honey processing by synthesis and secretion of saliva into nectar. The age structure of the colony is vital for increased production efficiency. This data reveals the molecular underpinning of SGs to accomplish their biological missions and provides new knowledge for the beekeeping industry for enhancing the management and production efficiency of the colony and honey quality through manipulation of potential target proteins. Biological significance: This study comprehensively analyses the characteristic of the proteome and phosphoproteome of honeybee salivary glands (SGs) between normal and single-cohort honeybee colonies. The SGs need a wide spectrum of proteins to support their multifaceted functions and ensure normal social management of the colony. The cephalic SG triggers labor transition from in-hive work to foraging behavior via the regulation of juvenile hormone and ethyl oleate titer. The thoracic SG stronger expressed of proteins related to carbohydrate and energy metabolism, protein folding, protein metabolism, cellular homeostasis and cytoskeleton to support the gland to efficiently enhance honey processing by synthesis and secretion of saliva into nectar. This data reveals the molecular underpinning of SGs to accomplish their biological missions and provides new knowledge for the beekeeping industry for enhancing the maintenance and production efficiency of the colony and honey quality through manipulation of potential target proteins. [Copyright &y& Elsevier]

Abstract: The development of the last day embryo to the first instar larva is an essential process in the honeybee life cycle. However, the molecular mechanism of this life transition is still unknown. The proteome and phosphoproteome of last day embryos (72h) and first instar larvae (24h, post hatching) were analyzed using 2-DE, multiplex fluorescent staining, mass spectrometry, bioinformatics, and qRT-PCR. Sixty-five proteins and 34 phosphoproteins changed their expression across the shift of embryos to larvae. The embryo stronger expression of proteins related to energy metabolism, development and amino acid metabolism suggests its high metabolic energy demand during active embryogenesis. While, the newly hatched larvae escalated the expression of proteins related to cytoskeleton, biosynthesis, protein folding, fatty acid and oxidative metabolism, particularly the higher phosphorylation of cytoskeleton and biosynthesis indicates their roles to ensure the fast growing larvae. These differences in protein expression level illustrate that specific protein functions are restricted to particular developmental stage. Our data suggest the essential changes of proteome and phosphoproteome to trigger the transition of embryo to larvae. This unravels the molecular event behind the first life cycle transition of honeybees and is potentially helpful for future reverse genetic studies in this model insect. [Copyright &y& Elsevier]

Abstract: To understand the olfactory mechanism of honeybee antennae in detecting specific volatile compounds in the atmosphere, antennal proteome differences of drone, worker and queen were compared using 2-DE, mass spectrometry and bioinformatics. Therefore, 107 proteins were altered their expressions in the antennae of drone, worker and queen bees. There were 54, 21 and 32 up-regulated proteins in the antennae of drone, worker and queen, respectively. Proteins upregulated in the drone antennae were involved in fatty acid metabolism, antioxidation, carbohydrate metabolism and energy production, protein folding and cytoskeleton. Proteins upregulated in the antennae of worker and queen bees were related to carbohydrate metabolism and energy production while molecular transporters were upregulated in the queen antennae. Our results explain the role played by the antennae of drone is to aid in perceiving the queen sexual pheromones, in the worker antennae to assist for food search and social communication and in the queen antennae to help pheromone communication with the worker and the drone during the mating flight. This first proteomic study significantly extends our understanding of honeybee olfactory activities and the possible mechanisms played by the antennae in response to various environmental, social, biological and biochemical signals. [Copyright &y& Elsevier]