T. Kubo, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: + 81 3 5684 2973, Tel.: + 81 3 5841 4821, E-mail: firstname.lastname@example.org
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 α-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 melanogasterα-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.
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 post-eclosion) 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 . We also purified a major 70-kDa protein that is present only in the forager-bee gland and identified it as an α-glucosidase . The gene for this enzyme is expressed specifically in the forager-bee gland .
In situ hybridization analysis revealed that the same type of secretory cell expresses the genes for bee-milk proteins in the nurse-bee gland and the gene for α-glucosidase in the forager-bee gland . Thus, the hypopharyngeal gland cell seems to enter two distinct states, which can be characterized by the pattern of protein expression, according to the age-dependent role change of the worker.
In the present study we characterized more precisely the cellular activity of the hypopharyngeal gland associated with the age-dependent role change by searching for genes that are expressed in the forager-bee gland but not in the nurse-bee gland. We isolated cDNAs for two carbohydrate-metabolizing enzymes (glucose oxidase and amylase), and showed that mRNAs for these enzymes were expressed specifically in the forager-bee gland. These results lend further support to the hypothesis that the function of the hypopharyngeal gland of the forager bee is highly specialized, expressing the genes for carbohydrate-metabolizing enzymes that are needed to process nectar into honey.
Materials and methods
European honeybees (A. mellifera L.) kept at the University of Tokyo were used. Nurse bees were collected when they were feeding the brood, and forager bees were collected when they returned to the colony after foraging for nectar and pollen, as described previously . The bees were anaesthetized by cooling on ice, and the hypopharyngeal glands were removed under a binocular microscope.
Cloning and sequencing of forager bee-specific cDNAs
Total RNA was extracted from the hypopharyngeal glands of nurse and forager bees and cDNAs were prepared by reverse transcription/PCR. Sequences common to forager and nurse bees were then removed from the forager-bee cDNA preparation by the cDNA subtraction method of Diatchenko et al. , using a commercially available kit (PCR-Select cDNA Subtraction kit, Clontech).
Partial sequencing of these clones revealed that one of them had significant sequence similarity with the α-amylase genes of many other animals. A full-length clone was obtained by screening a cDNA library constructed using RNAs from the hypopharyngeal glands of worker honeybees  by plaque hybridization using the partial cDNA as the probe, according to a standard protocol .
For sequencing, pBluescript containing the insert was prepared by the method of Short et al. . Various deletion derivatives of the DNA fragment, prepared using exonuclease III and mung bean nuclease , were sequenced by the dideoxy chain-termination method of Sanger et al.  using an ABI 377 DNA sequencer and a Taq Dye terminator cycle sequencing kit (Applied Biosystems). The nucleotide sequences of both strands were determined.
Detection of hypopharyngeal gland mRNA by Northern blotting
Total RNAs (1 µg) extracted from the hypopharyngeal glands of nurse and forager bees were separated by 1.2% formaldehyde/agarose gel electrophoresis, transferred to a nylon membrane (NEN) and cross-linked by UV irradiation. The RNA on the filter was hybridized with a [32P]-labelled probe for 12 h at 42 °C and then the filter was washed with 2 × NaCl/Cit followed by 0.5 × NaCl/Cit (1 × NaCl/Cit = 150 mm sodium chloride, 15 mm sodium citrate, pH 7.0) for 15 min each at room temperature. The hybridization pattern was recorded by exposing the filter to X-ray film and then the probe was removed by washing the filter in Tris/EDTA (10 mm Tris/HCl pH 8.0, 1 mm EDTA) containing 0.5% SDS at 90–100 °C for 10 min. A second probe was then hybridized to the same filter. The probes used were PCR products corresponding to +28 to +1915 of the glucose oxidase cDNA, and +423 to +759 of the amylase cDNA.
Assay of glucose oxidase and amylase activities
Glucose oxidase activity was assayed by the method of White et al. . The reaction mixture was 0.1 m sodium-phosphate buffer, pH 6.1, containing 1.5 m glucose as the substrate and 0.1 mg·mL−1o-dianisidine. To determine the amount of H2O2 liberated from the glucose, 1 µL of 0.04 µg·mL−1 peroxidase and 10 µL of test sample was added to the reaction mixture, bringing the total volume to 171 µL. The solution was incubated for 60 min at 37 °C and then the reaction was halted by adding 10 µL of 1 m HCl, and the absorbance at 400 nm (oxidized o-dianisidine) was measured using a spectrometer.
Amylase activity was assayed by a dinitrosalicylic acid procedure . The assay was performed in a total volume of 100 µL of 0.1 m sodium phosphate buffer, pH 6.1, containing 1% soluble starch as the substrate. After incubation for 60 min at 37 °C, an equal volume of dinitrosalicylic acid was added to stop the reaction. To determine the amount of glucose liberated the solution was boiled for 5 min and then diluted 10-fold with water. The absorbance was measured at 550 nm using a spectrometer.
Purification of amylase and glucose oxidase
Hypopharyngeal glands collected from about 500 forager bees were homogenized in buffered insect saline (10 mm Tris/HCl, pH 7.4, containing 130 mm NaCl, 5 mm KCl, and 1 mm CaCl2) containing 1 mm phenylmethanesulfonyl fluoride, 0.1 µg·mL−1 pepstatin and 100 µg·mL−1 leupeptin in a glass homogenizer on ice. The homogenate was centrifuged at 13000 g for 20 min. The supernatant (4 mL) was diluted with 10 mm Tris/HCl, pH 7.4, up to 52 mL. The proteins were separated by fast protein liquid chromatography on an anion-exchange (Mono Q) column equilibrated with 10 mm Tris/HCl, pH 7.4 containing 10 mm NaCl. The column was developed with a linear gradient of 10 mm–1.0 m NaCl. Amylase activity was detected in the flow-through fraction. Glucose oxidase activity eluted from the column as a single peak at about 200 mm NaCl.
One-quarter of the fractions containing glucose oxidase activity were diluted fourfold with 10 mm Tris/HCl, pH 7.4, and loaded onto a cation-exchange (Mono S) column equilibrated with 10 mm Tris/HCl, pH 7.4, containing 50 mm NaCl. The column was developed with a linear gradient of 50–400 mm NaCl. Glucose oxidase activity eluted as a single peak at 290 mm NaCl.
The flow-through fraction from the Mono Q column was loaded onto a Mono S column equilibrated with 10 mm Tris/HCl, pH 7.4, containing 10 mm NaCl. The column was developed with a linear gradient of 10–150 mm NaCl. Amylase activity eluted as a single peak at 45 mm NaCl.
Determination of the N-terminal sequences of amylase and glucose oxidase
The partially purified enzymes (active Mono Q fractions, about 50 µg each) were separated by SDS-PAGE  and blotted electrophoretically onto nylon membranes. The sections of the membranes that contained the proteins were the excised and placed directly into an automated protein sequencer (Shimazu).
Characterization of the amylase and glucose oxidase genes by Southern blotting
Aliquots of genomic DNA (10 µg each) extracted from the heads and thoraxes of the worker honeybees were each digested with one of several restriction endonucleases. The DNA fragments were separated by 0.8% agarose gel electrophoresis, and then transferred to a nylon membrane (NEN). The DNA on the membrane was hybridized with a [32P]-labelled cDNA probe for 12 h at 65 °C and then washed with 2 × NaCl/Cit followed by 0.5 × NaCl/Cit for 15 min each at room temperature. After autoradiography the probe was removed from the filter by washing in Tris/EDTA (10 mm Tris/HCl pH 8.0, 1 mm EDTA) containing 0.5% SDS at 90–100 °C for 10 min. Then the same membrane was incubated with a second probe. The probes used were PCR products corresponding to nucleotides +9 to +213 of the glucose oxidase cDNA, and +34 to +348 of the α-amylase cDNA.
Isolation and analysis of cDNAs for putative Apis amylase and glucose oxidase
To characterize the function of the forager-bee’s hypopharyngeal gland more precisely, we tried to isolate genes that are expressed in the forager-bee gland but not in the nurse-bee gland by the cDNA subtraction method. By removing the nurse-bee gland cDNAs from the forager-bee gland cDNAs we obtained a few candidate cDNA clones, one of which had significant sequence similarity to the α-amylase genes of many other animals. To isolate a full-length clone of the putative honeybee amylase, we screened a cDNA library of the hypopharyngeal gland of the worker by the colony hybridization method using the partial cDNA as the probe. By screening 20 000 clones, we obtained 40 positive clones. Five positive clones arbitrarily selected from the 40 positive clones had identical nucleotide sequences for each 300 bp from the 5′ and 3′ ends.
One of these clones (pA151) was sequenced and found to encode a protein consisting of 493 amino acid residues (the nucleotide sequence of pA151 has been deposited in the DDBJ, EMBL and GenBank sequence data banks and is available under the accession number AB022907). The hydropathy profile showed that 17 residues starting from the first methionine were hydrophobic, suggesting that the sequence is a signal peptide (data not shown). We searched for proteins that exhibit significant sequence similarity to the protein encoded by pA151 in the PIR, SWISS-PROT, and PRF protein sequence databases and we found 60.5% sequence identity between this protein and the proteins encoded by the fruit fly (Drosophila melanogaster) α-amylase precursor gene (Fig. 1), strongly suggesting that the clone is a cDNA for an Apisα-amylase homologue.
During the screening of the putative Apis amylase cDNAs, we found that one positive clone (pA150) contained a glucose dehydrogenase-like sequence artificially attached to the putative amylase sequence. This clone had formed during the construction of the cDNA library because both sequences had the adapter sequence of the library at their 5′ end. As glucose oxidase activity has been detected in honey  and is thought to be derived from the hypopharyngeal gland of the forager bee , we thought it probable that this sequence encoded Apis glucose oxidase. Therefore, we screened a cDNA library of the worker-bee hypopharyngeal gland using this sequence as a probe; we isolated a full-length clone (pG61). This cDNA encodes a protein consisting of 615 amino acid residues (Accession number AB022908). The protein showed the highest sequence similarity (44.4%) with glucose dehydrogenase of D. pseudoobscura followed by glucose oxidase of Aspergillus niger (23.8%) (Fig. 2). However we concluded that the clone encodes an Apis glucose oxidase homologue based on the results of protein sequencing.
Purification of glucose oxidase and amylase from the hypopharyngeal gland
To examine whether amylase and glucose oxidase are actually expressed in the forager-bee gland, we tried to purify both enzymes from the homogenate of the hypopharyngeal glands of the forager bee. When the homogenate of the hypopharyngeal gland was loaded onto a Mono Q column, amylase activity was detected in the flow-through fraction and glucose oxidase activity eluted from the column as a single peak at 120 mm NaCl. The active fractions were each pooled and loaded onto a Mono S column. Each enzyme eluted as a single peak coinciding with a peak of protein (Figs 3A and 4A).
Single bands with molecular masses of 57 kDa and 85 kDa were detected when the active fractions for amylase and glucose oxidase were subjected to SDS/PAGE (Figs 3B and 4B), indicating that these enzymes were purified to near homogeneity. As summarized in Tables 1 and 2 the specific activities of the purified amylase and glucose oxidase preparations were higher by 42- and 34-fold than those of crude homogenate, respectively, suggesting that these enzymes are relatively abundant in the forager-bee gland.
Table 1. Purification of Apisα-amylase.
Specific activity (U·mg−1)
Table 2. Purification of Apis glucose oxidase.
Specific activity (U·mg−1)
To examine whether these proteins were actually encoded by the cDNAs we had isolated we sequenced the N-terminal region of each protein. Nineteen N-terminal amino acids of α-amylase were sequenced and corresponded to Glu18 to Leu36 of the putative protein encoded by pA151 (Fig. 1), indicating that this clone is a cDNA for Apisα-amylase. The 17 residues starting from the first methionine were thought to be signal sequence, which is consistent with the hydropathy profile. The predicted molecular mass of the mature amylase, without the signal sequence, was 53 kDa, which agrees well with that of the purified amylase determined by SDS/PAGE (57 kDa).
Fourteen N-terminal amino acids of glucose oxidase were sequenced and corresponded to Ala2 to Gln15 of the putative protein encoded by the cDNA (Fig. 2), which clearly indicates that the clone pG61 is a cDNA for Apis glucose oxidase, and not for glucose dehydrogenase, even though the protein encoded by pG61 had a higher sequence similarity (44.4%) with Drosophila glucose dehydrogenase . The predicted molecular mass of the mature glucose oxidase, 68 kDa, suggests that it undergoes N-linked glycosylation as does the glucose oxidase of A. niger.
Expression of mRNAs for amylase and glucose oxidase before and after the age-dependent role change
Previously, we showed that gene expression in the hypopharyngeal gland cell changes from bee-milk protein to α-glucosidase in accordance with the age-dependent role change [11,14]. Therefore, we examined whether the expression of the genes for amylase and glucose oxidase also changes in accordance with the role change by Northern blotting using the corresponding cDNAs as probes. mRNAs for both amylase and glucose oxidase were detected in the hypopharyngeal gland of the forager bee and neither was detected in that of the nurse bee (Fig. 5). Thus, the hypopharyngeal gland of the forager bee expresses at least three genes for carbohydrate-metabolizing enzymes.
Characterization of honeybee amylase and glucose oxidase genes
To determine the copy numbers of the genes for Apis amylase and glucose oxidase, Southern blotting was performed using Apis genomic DNA digested with various restriction enzymes and the corresponding cDNAs as probes. As for the amylase gene, two or three bands were detected in samples digested with DraII, EcoRV or HindIII, and a single band was detected in samples digested with BglII, EcoRI or XhoI (Fig. 6A). The probe DNA used for Southern blotting did not contain any sites for these enzymes, so the amylase gene appears to be a multicopy gene or a single gene having an intron(s). By contrast, a single band was detected by the probe for glucose oxidase irrespective of the restriction enzyme used, strongly suggesting that there is only a single copy of the gene in the Apis genome (Fig. 6B).
From the results reported in this paper and those reported previously [10–14], it is clear that the hypopharyngeal gland cell of the forager bee expresses at least three genes for carbohydrate-metabolizing enzymes, α-glucosidase, amylase and glucose oxidase, that are not expressed by the hypopharyngeal gland cell of the nurse bee. Amylase is thought to be needed to convert starch of plant origin, which is found in nectar, into glucose, and glucose oxidase is needed to convert glucose to gluconic acid and hydrogen peroxide [5,20]. The gluconic acid keeps the honey acidic and, together with hydrogen peroxide, it has an antiseptic action. Thus, these enzymes are essential for the forager bee’s task of converting nectar into honey. The Apis glucose oxidase did not contain a putative signal sequence; it appears to be secreted without the aid of a signal sequence.
Previously we showed that α-glucosidase is the major protein present in the hypopharyngeal gland of the forager bee, reaching about 50% of the total protein of the gland . Now we estimate that amylase and glucose oxidase each account for ≈ 2–3% of the total protein, based on our purification results. Taken together, our results lend further support to the notion that the hypopharyngeal gland cell function of the forager bee is highly specialized, expressing specifically the genes for enzymes that are needed for honey production. At present, it is uncertain whether each secretary cell expresses the genes for all of these enzymes.
Northern blotting showed that the genes for these three carbohydrate-metabolizing enzymes are expressed in the hypopharyngeal gland according to the age-dependent role change of the worker bee. Whether or not a common signal transduction pathway activates all three genes in the hypopharyngeal gland will be an interesting topic for future research.
Our Southern blotting results suggest that the amylase gene is either a multicopy gene or a single gene with an intron(s). Amylase is thought to be expressed and secreted the salivary gland, gut and other digestive organs as well as by the hypopharyngeal gland, so regulation of the expression of the amylase gene(s) may be complex. By contrast, we have shown that the glucose oxidase gene is a single-copy gene. Considering that the N-terminal sequence of the purified glucose oxidase was encoded by the clone pG61 and that the gene for glucose oxidase is single, it is certain that pG61 encodes an Apis glucose oxidase and not other structurally related proteins, such as glucose dehydrogenase. Glucose oxidase is very rare in the animal kingdom , and, to our knowledge, this is the first report to describe the purification and cDNA cloning of a glucose dehydrogenase of animal origin. Analysis of the homologues of this enzyme in other insect species would be interesting from the perspective of comparative molecular biology.
This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Cooperation (JST).
Enzymes: glucose oxidase (EC 22.214.171.124); α-amylase (EC 126.96.36.199); exonuclease III (EC 188.8.131.52); mung bean nuclease (EC 184.108.40.206).Note: the nucleotide sequence data published here have been deposited in the DDBJ, EMBL and GenBank sequence data banks and are available under the accession numbers AB022907, for Apis mellifera mRNA for glucose oxidase (complete codons) and AB022908, for Apis mellifera mRNA for amylase (complete codons).