Worker honeybees change their behaviour from the role of nurse to that of forager with age. We have isolated cDNA clones for two honeybee (Apis mellifera L.) genes, encoding a-amylase and glucose oxidase homologues, that are expressed in the hypopharyngeal gland of forager bees. The predicted amino acid sequence of the putative Apis amylase showed 60.5% identity with Drosophila melanogaster a-amylase, whereas that of Apis glucose oxidase showed 23.8% identity with Aspergillus niger glucose oxidase. To determine whether the isolated cDNAs actually encode these enzymes, we purified amylase and glucose oxidase from homogenized forager-bee hypopharyngeal glands. We sequenced the N-terminal regions of the purified enzymes and found that they matched the corresponding cDNAs. mRNAs for both enzymes were detected by Northern blotting in the hypopharyngeal gland of the forager bee but not in the nurse-bee gland. These results clearly indicate that expression of the genes for these carbohydrate-metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age-dependent role change of the worker.Keywords: amylase; behaviour; gene expression; glucose oxidase; honeybee.The honeybee (Apis mellifera L.) is a social insect and its colony is composed of a queen, drones, and workers. Workers perform almost all of the tasks in the colony except egg laying, but the tasks performed by an individual worker change depending on age after eclosion (age polyethism). The life-span of a worker bee is usually 30±40 days. Young workers (nurse bees, generally less than 14 days posteclosion) take care of their brood by synthesizing and secreting bee milk (royal jelly), whereas older workers (foragers, more than 10 days posteclosion) forage for nectar and process it into honey. They also have an intervening period of work in the hive devoted to other duties such as comb-building (days 10±27) [1±5]. In parallel with this age-dependent role change, physiological changes occur in certain organs of the worker. For example, the hypopharyngeal gland, which is believed to synthesize bee milk [6±8], is well developed in the nurse bee but shrinks in the forager, which then develops the ability to hydrolyse the sucrose of nectar into glucose and fructose [4,9].Previously, we purified three major proteins (of 50, 56 and 64 kDa) from homogenates of nurse-bee glands, and identified them as bee-milk proteins [10,11]. cDNA cloning revealed that the 50-and 64-kDa proteins (identical to RJP57-1) and RJP57-2, reported by Klaudiny et al. [12,13], are structurally related. mRNA for the 64-kDa protein is present in the nurse-bee gland but not in the forager-bee gland. By contrast, mRNA for the 56-kDa protein is present in the glands of both nurse and forager bees although the 56-kDa protein is present only in the nurse-bee gland, suggesting that expression of the 56-kDa protein is regulated at the translational level [11]. We also purified a major 70-kDa protein that is present only in the forager-bee gland ...

Major proteins synthesized in the hypopharyngeal gland of the worker honeybee change from bee-milk proteins to alpha-glucosidase in accordance with the age-dependent role change of the worker bee. Previously, we showed that the gene for alpha-glucosidase is expressed specifically in the forager-bee gland [Ohashi, K., Sawata, M., Takeuchi, H., Natori, S. & Kubo, T. (1996) Biochem. Biophys. Res. Commun. 221, 380-385]. Here, we describe the isolation and analysis of cDNAs for two bee-milk 56-kDa and 64-kDa proteins. The 56-kDa protein was a glycoprotein which shared 63.2% and 56.9% amino acid sequence identities with proteins encoded by cDNA for royal-jelly-related protein 57-1 (pRJP57-1) and pRJP57-2. The 64-kDa protein cDNA was identical to pRJP57-1. Thus, these bee-milk proteins seem to form a structurally related protein family. The gene for the 64-kDa protein/RJP57-1 was expressed specifically in the nurse-bee gland, whereas that for the 56-kDa protein was expressed in both the nurse-bee and forager-bee glands. mRNAs for the 56-kDa and 64-kDa proteins were detected by in situ hybridization in a whole acinus of the nurse-bee gland, whereas mRNAs for the 56-kDa protein and alpha-glucosidase were detected in that of the forager-bee gland. Therefore, the individual secretory cells of the acinus of the hypopharyngeal gland were shown to express these genes differently with the age-dependent role change of the worker bee.

The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis using affinity-purified antibodies against three major proteins (50, 56, and 64 kDa) of the nurse-bee gland showed that they are synthesized selectively and secreted as bee-milk proteins. Immunofluorescence study showed that the proteins condense in the duct after secretion from acini. However, a major 70-kDa protein synthesized specifically in the forager-bee hypopharyngeal gland was identified as an alpha-glucosidase. Therefore, the hypopharyngeal gland seems to have two distinct states differentiated by synthesizing of different major proteins depending on the age-dependent role change.

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A growth factor from the conditioned medium of NIH-Sape-4, an embryonic cell line of the flesh fly, was purified to homogeneity. This growth factor, termed IDGF, stimulated the proliferation of NIH-Sape-4 cells in an autocrine manner; it was a homodimer of a protein with a molecular mass of 52 kDa, and its specific activity was comparable with those of mammalian growth factors. Immunoblotting experiments revealed that unfertilized mature eggs of the flesh fly contained this growth factor, a certain level of which was maintained throughout embryonic development. Analysis of cDNA for this growth factor showed that this factor is a novel protein consisting of 553 amino acid residues. No significant sequence similarity was found between this factor and other proteins except atrial gland granule-specific antigen of Aplysia californica.

Mushroom bodies (MBs) are considered to be involved in higher-order sensory processing in the insect brain. To identify the genes involved in the intrinsic function of the honeybee MBs, we searched for genes preferentially expressed therein, using the differential display method. Here we report a novel gene encoding a putative transcription factor (Mblk-1) expressed preferentially in one of two types of intrinsic MB neurones, the large-type Kenyon cells, which makes Mblk-1 a candidate gene involved in the advanced behaviours of honeybees. A putative DNA binding motif of Mblk-1 had significant sequence homology with those encoded by genes from various animal species, suggesting that the functions of these proteins in neural cells are conserved among the animal kingdom.

The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.

We have previously used the differential display method to identify a gene that is expressed preferentially in the mushroom bodies of worker honeybees and to show that it encodes a putative inositol 1,4,5‐trisphosphate receptor (IP3R) homologue (Kamikouchi et al. [1998] Biochem. Biophys. Res. Commun. 242:181–186). In the present study, we examined whether the expression of some of the genes for proteins involved in the intracellular Ca2+ signal transduction is also concentrated in the mushroom bodies of the honeybee by isolating cDNA fragments that encode the Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and protein kinase C (PKC) homologues of the honeybee. In situ hybridization analysis revealed that the expression of these genes was also concentrated in the mushroom bodies of the honeybee brain: The CaMKII gene was expressed preferentially in the large‐type Kenyon cells of the mushroom bodies, whereas that for PKC was expressed in both the large and small types of Kenyon cells. The expression of the genes for IP3R and CaMKII was concentrated in the mushroom bodies of the queen and drone as well as in those of the worker bee. Furthermore, the enzymatic activities of CaMKII and PKC were found to be higher in the mushroom bodies/central bodies than in the optic and antennal lobes of the worker bee brain. These results suggest that the function of the intracellular Ca2+ signal transduction is enhanced in Kenyon cells in comparison to other neuronal cell types in the honeybee brain. J. Comp. Neurol. 417:501–510, 2000. © 2000 Wiley‐Liss, Inc.