EXPRESSION OF ALPHA-GLUCOSIDASE GENE IN

HYPOPHARYNGEAL GLANDS OF EASTERN HONEYBEE

WORKER Apis cerana indica

C h a n p e n C h a n c h a o1

, P r a p a i p i t S r i m a w o n g2

,

S i r i w a t W o n g s i r i1

1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.2Research center, Ramathibodi Hospital, Bangkok, Thailand. E-mail: chanpen@sc.chula.ac.th

Received 29 November 2005; accepted 04 October 2006

S u m m a r y

Duties of worker bees in a hive change according to their age. Temporal and spatial expres-

sion of developmentally regulated genes relating to this is interesting. In order to control the age

of Apis cerana indica, emerged bees were marked with painting color and counted as 0 day.

Bees at desired ages were collected during 0-29 days with the time interval of 3 days. The re-

sults showed that hypopharyngeal glands (hpg) change their size from large to shrunken and

their colour from creamy to pale. The largest acini were found in worker bees 15 to 18 days old

and the smallest in 29 days old. Alpha-glucosidase (ag) activity assay in crude extract was low

in emerged bees. Then, the activity increased in workers 18 days old and got the highest in those

24 days old. Activity staining of crude extract by native polyacrylamide gel, ag was low from

18-day and high from 29-day old worker bees. In addition, the expression profile of ag was ob-

tained by RT-PCR. It indicates that the expression of ag was high in foragers. The obtained

cDNA sequence shows closely relationship (82.81% similarity) to the cDNA in A. mellifera.

Keywords: Apis cerana indica, alpha-glucosidase, development, expression, nurse bee,

forager, activity, RT-PCR, cDNA, phylogeny.

INTRODUCTION

Honeybee colonies as highly eusocial

insects are composed of 3 castes: a female

queen, female workers, and male drones.

Most of the population in a colony are

workers responsible for all tasks such as

brood rearing, wax secreting and comb

building, food handling and storing, pollen

or nectar foraging, and a community de-

fense. A division of labor in honeybee col-

onies is based on worker age. The workers

change jobs when their age changes. This

form of behavioral development is called

“age polyethism” (Huang et al. 1991).

Hypopharyngeal glands (hpg) located in

the heads of worker bees are paired long tu-

berous organs connected to many acini (se-

cretory glands). In A. mellifera, the acini of

emerged bees look plump and creamy. The

hpg synthesizes and secretes a substance

rich in protein, called bee milk, or royal

jelly (rj), which is fed to all larvae (Dade

1994). Later, hpg change to produce diges-

tive enzymes, including alpha-glucosidase

(ag), invertase, etc (Brouwers 1982). At

this stage, worker bees become foragers.

The ag is alpha-D-glucoside gluco-

hydrolase and belongs to EC 3.2.1.20. The

ag catalyzes the hydrolysis of terminal,

non–reducing alpha-1, 4-linked glucose

residues from aryl-glucosides, disacchari-

des or oligosaccharides. Other common

names of ag are invertase, sucrase, and

maltase.

Different proteins from hpg of

A. mellifera at different ages were reported.

Three major proteins with molecular

masses of 50, 56, and 64 kDa were restric-

Vol. 50 No. 2 2006 Journal of Apicultural Science 5

tively found in nurse bees, whereas a major

70-kDa protein was specifically found in

foragers. Immunoblotting analysis against

50, 56, and 64 kDa proteins confirmed that

they were only detected in hpg of nurse

bees and also existent in rj. It suggested

that these proteins were synthesized in hpg

of nurse bees and secreted as constituents

of rj. In addition, a 70 kDa protein was pu-

rified and immunoblotted, this protein was

positively detected in hpg of foragers only.

Subsequently, the 70 kDa protein was char-

acterized and identified as ag (Kubo et al.

1996).

Recently, proteins at molecular masses

of 57 and 85 kDa were also purified from

hpg of A. mellifera foragers and determined

by Sodium Dodecyl Sulfate Polyacryla-

mide Gel Electrophoresis (SDS PAGE).

These proteins were already characterized

as amylase and glucose oxidase, respec-

tively (Ohash i et al. 1999). They are

members of the sequence-related family of

alpha-glycohydrolases (family 13) contain-

ing many important digestive enzymes

(Yao et al., 2003).

Huber and Mathison (1976) purified ag

from whole bodies of honeybees. The

A. mellifera ag, Mr 82,000, is found in the

head and abdomen of honeybees and its

properties are similar to that of ag found in

honey. Thus, this enzyme is probably syn-

thesized in hpg and converts nectar sucrose

into glucose and fructose in the crop. The

pH optimum of each honey bee sucrase

was different from each other according to

the substrates as sucrose (pH 5.5) and

p-nitrophenyl-alpha-glucoside (pH 6.5).

Takewaki et al. (1980 and 1993) re-

ported the purification and some properties

of both agI and agII from adult honey bees.

Two kinds of the enzymes could be

chromatographically separated according

to their solubility in ammonium sulfate.

The molecular mass of agI and agII was

estimated to be approximately 98 and

76 kDa, respectively, by SDS disc electro-

phoresis. Their pH optimum was 5.0. Both

ag readily hydrolysed phenyl-alpha-

glucoside, sucrose and maltose.

Different proteins from worker bees at

different stages indicate the differential

gene expression. That includes the ag gene

also.

In A. mellifera, the mRNA for the ag

was detected, cloned, and characterized.

The deduced amino acid sequence of 650

residues revealed 41.9% identity to maltase

of mosquito (Aedes aegypti) and 42.2%,

46.3% and 46.2% of maltase 1, 2, and 3 of

fruit fly (Drosophila melonogaster), re-

spectively (Ohashi et al. 1996).

According to the data mentioned above,

a lot of researches have been done in

A. mellifera, western bee, but not in

A. cerana indica which is native and eco-

nomic to Thailand. This research is in-

volved in the relationship of size of hpg

and expression of ag in hpg. Specific activ-

ity of ag in crude extract and RT-PCR were

used to analyse the function of ag. Finally,

a phylogenetic tree of ag was constructed

in order to reveal the homology of the gene

in other organisms.

MATERIALS AND METHODS

Sample collection

Colonies of Apis cerana indica were

taken from Nakhon Sri Thammarat provin-

ce and maintained at Nonthaburi province.

Newly emerged workers were marked on

their thorax with paint markers (water-

proof) and introduced into a free-flying

colony. The colony was fed with sugar

candy everyday. Thereafter, marked bees

were collected for 29 days with the time in-

terval of 3 days and were immediately

stored at -20°C until use. For RNA extrac-

tion, nurse bees were collected while they

were feeding brood. Forager bees were col-

lected when they returned to the colony af-

ter foraging for nectar and pollen. Samples

were stored in liquid N2 until used.

6

Measurement of hpg

Dissected hpg were stored in 0.85%

(w/v) NaCl solution. The shortest (a) and

the longest (b) diameters (in µm) of acini

were measured by an ocular scale in light

microscope. The area of hpg was evaluted

as S (µm2) = π × a/2 × b/2 (Sasagawa et al.

1989).

Enzyme assay

The ag activity was determined by mea-

suring glucose liberated from sucrose. The

method was modified from Momose’s

method (Kubo et al. 1996). Twenty hpg

were dissected and stored in 500 µl buffer

insect saline (10 mM Tris, 130 mM NaCl, 5

mM KCl, and 1 mM CaCl2, pH 7.4) con-

taining 1 mM phenylmethylsulfonyl-

fluoride (PMSF), 0.1 µg/ml pepstatin, and

100 µg/ml leupeptin. The glands were ho-

mogenized and centrifuged 2 times at 4°C,

700 × g for 10 min. The supernatant was

stored at -20°C. Activity staining for ag

was modified from Ta n i m u r a et al.

(1979). Briefly, after native PAGE, the gel

was incubated in 10 mM sodium acetate

buffer containing 0.5 M sucrose (pH 5.0) at

45°C for 30 min. After washed, the gel was

boiled in freshly prepared solution of 0.1%

(w/v) triphenyltetrazolium chloride in

0.5 N NaOH for 3 min. The positive band

of ag on the gel appeared in red.

Primer design

Primers of ag in A. cerana for RT-PCR

were designed from cDNA sequences of ag

in A. mellifera (Ohashi et al. 1996, acces-

sion: D79208). Forward primers (FW) are

5’-tcgac ttcta gttgg tagca tgaag g-3’ (FW1),

5’-gctta tcgag gcata cacga-3’ (FW2), and

5’-acgag gaaca aatcg tggat-3’ (FW3), re-

spectively. Also, reverse primers (R) are

5’-ctagt cagtg ctgca catga gaaag g-3’ (R1),

5’-gacgt acatg ccacc aagtg-3’ (R2), and

5’-gtcta ttctt tgaag cggcg-3’ (R3), respec-

tively. In addition, control primers were de-

signed from 28s RNA in A. mellifera (FW:

5’-aaaga tcgaa tgggg atatt c-3’ and R:

5’-caccg ggtcc gtacc tcc-3’) and elongation

factor (ef) genes in A. cerana (FW: 5’-tcgct

tttac tcttg gtgtg a-3’ and R: 5’-aaact cccaa

catat tatct cca-3’).

RT-PCR

Total RNA (200 ng) of emerged bees,

nurse bees 6 days old, and foragers were

isolated from hpg by SV total RNA isola-

tion kit (Promega, catalog# Z3100).

RT-PCR was performed according to ac-

cess RT-PCR system (Promega, catalog#

A1250). The RT-PCR cycling profile of ag

was modified from Ohashi et al. (1996

and 1997). As a control, 28s RNA and ef

primers were used as RNA reference mar-

kers. The PCR amplification was at the

same condition as that of ag primers.

Sequence alignment

Obtained RT-PCR product was purified

by QIAquick PCR purification kit (Qiagen,

catalog# 28104) and directly sequenced by

an automated sequencer (ABI prism, model

377). The nucleotide and deduced amino

acid sequences were aligned by using

Genetyx program. The cDNA sequences

were aligned and compared to the se-

quences of maltase 1 in A. mellifera

(XM_393379), Drosophila melanogaster

CG14934-PA (NM_135678), sucrose spe-

cific enzyme II of the PTS (ScrA) gene in

Lactobacillus sakei (AF401046), and

Culicoides sonorensis clone CsMAL1

maltase (AY603565).

Statistical analysis

ANOVA was applied and multiple range

test was used to detect significant differ-

ences between the means at p < 0.05.

RESULTS

Hypopharyngeal gland size and age

A tendency is visible that the size of

hypopharyngeal glands (hpg) increases

during day 0 to 15 after emerging of

worker bees. The size of hpg of day 0 and

of day 3 is significantly smaller than the

size of hpg of day 15, while the size of hpg

of day 15 to day 18 is not significantly dif-

Vol. 50 No. 2 2006 Journal of Apicultural Science 7

ferent (Fig. 1). After day 18, the size of hpg

is like to decrease. The size of hpg of day

24, 27 and 29 is significantly smaller, than

the size of hpg of day 15. This coincides to

the task of worker bees. Considering the

overall tendency of size changes, while the

increasing size of acini (day 0 to 15) is ob-

served, bees become nurse bees. After the

size of acini decreases (day 15 to 29), bees

become foragers.

Activity of ag

The ag activity examined from crude

extract of hpg at various ages, from day 0

to day 29, and computed by comparing to

the standard curve of glucose is shown in

Fig. 2. Although specific activity of all

stages is not significantly different by using

One-way ANOVA, the tendency of chang-

ing activity is still visible. It shows that the

specific activity of ag from day 0 (0.0239

unit/mg protein) to day 18 (6.7363

units/mg protein) is lower, than the specific

activity from day 24 (9.0811 units/mg pro-

tein). Although the specific activity of ag

from day 21 (3.4024 units/mg protein) is

low, it is still possible to assume that the

tendency of ag specific activity increased

during the period. After day 24, both de-

crease, on day 27 (5.8187 units/mg protein)

and on day 29 (3.5738 units/mg protein).

By comparing nurse bees (d. 15) and

foragers (d. 24), presented in figures 1 and

2, it is visible that the size of acini de-

creased while the ag activity increased. The

size of acini of bees 24 days old is signifi-

cantly smaller than that size of bees 15

days old. In contrast, the ag activity is still

very low in nurse bees 15 days old. Then, it

is higher in foragers 24 days old.

Activity stain of ag

The ag activity on native polyacryla-

mide gel appeared in red. Only one activity

band was visible. Fig. 3 shows that higher

activity of ag could be detected on the lane

containing crude extract of forager bees

(lane 2) as compared to the ag activity of

crude extract of nurse bee (lane 1).

Fig. 3. Native polyacrylamide gel of ag

in crude extract. Detected by activity stain-

ing, a positive band is observed in 6-day

old worker bee (8 µg; lane 1) and forager

bee (8 µg; lane 2).

The expression level of ag by RT-PCR

8

Figure 1. The overall size (calculated area) of acini in worker bees at various ages. Ten acini at eachstage were measured. Error bars indicate standard deviation. Different letters indicate significantdifferences between means PMFs<0.05.

Fig. 4 shows that the expression of ag

was not detectable in emerged bees but was

detectable in worker bees (from 6-d.

worker bees to foragers). The expression of

ag was also high in foragers. As control,

the amplified product was obtained from

bees at any stages by using primers of 28S

RNA in A. mellifera (350 bp) and of elon-

gation factor (ef) in A. cerana (100 bp)

(data not shown).

The cDNA sequence of ag and its phylo-

genetic tree

According to RT-PCR, cDNA (1,549

bp) and deduced amino acid (516 residues),

the sequences of ag in A. cerana indica

was obtained (Fig. 5). The ag cDNA of

A. cerana indica was compared to that

from other organisms. The similarity be-

tween cDNA sequences was established us-

ing a program of nucleotide-nucleotide

BLAST (Basic Local Alignment Search

Tool of the National Center for Biotechnol-

ogy Information). The length of cDNA in

base pairs (bp) was from RT-PCR as men-

tioned in Materials and Methods. It is par-

tially similar to the ag in A. mellifera at

82.81% (1,557 bp comparison), to maltase

Vol. 50 No. 2 2006 Journal of Apicultural Science 9

Figure 3. Native polyacrylamide gel of ag incrude extract. Detected by activity staining, apositive band is observed in 6-day old workerbee (8 µg; lane 1) and forager bee (8 µg;lane 2).

Figure 2. The specific activity of ag in worker bees of different age. Error bars indicate standarddeviation. No significant differences between means PMFs<0.05 were determined but the tendencyof activity is visible.

10

Figure . The RT-PCR product (350 bp) of ag from bees at various age. Lane 1, 100 bp laddermarker; lane 2, negative control; lane 3–5, emerged bees; lane 6–8, 6-day old worker bees; lane9–11, nurse bees; and lane 12–14, forager bees.

Figure . Phylogenetic trees of ag by UPGMA (A) and NJ (B). Am malt stands for maltase 1 inA. mellifera. Dm cg stands for Drosophila melanogaster CG14934-PA. Ls scrA stands for sucrosespecific enzyme II of the PTS in Lactobacillus sakei. Cs malt stands for Culicoides sonorensis cloneCsMAL1 maltase. In addition, Ac agand Am agstand for alpha-glucosidase in A. cerana indica andin A. mellifera, respectively. Numbers on branches of both trees indicate genetic distance.

in A. mellifera at 53.44% (1,570 bp com-

parison), to maltase in Culicoides

sonorensis at 48.75% (1,600 bp compari-

son), to cg in Drosophila melanogaster at

53.66% (1,584 bp comparison), and to Su-

crose-specific enzyme II of the proton

transport system, PTS (ScrA) in

Lactobacillus sakei at 50.82% (1,159 bp

comparison).

Aligned sequences were imported into a

phylogenetic analysis program of Genetyx.

The ag cDNA sequence of A. mellifera (ac-

cession number: D79208 in GenBank) was

used as an outgroup. The patterns of ge-

netic distance from Unweighted Pair Group

Method with Arithmetic mean (UPGMA)

and Neighbor-joining (NJ) programs are

indicated as shown in Fig. 5. The trees

share similar characters, which show that

the ag sequences in A. cerana indica and in

A. mellifera are closedly similar. According

to both trees, genetic distance of ag be-

tween both organisms are very small.

DISCUSSION AND CONCLUSIONS

Hypopharyngeal glands (hpg) of Apis

cerana indica were well developed in nurse

bees, however, they were shrunk in forager

bees older than 27 days (Fig. 1). The size of

the glands should be related to the duty of

nurse bees because bees at this stage have

to feed royal jelly (rj) to brood and a queen.

The hpg are known to be responsible for

synthesizing rj. The size of the acini in-

creased significantly between day 3 and

day 15 after emerging of the bees. After

day 18, the size decreased significantly

(Fig. 1).

The ag specific activity is high from

worker bees 18 to 29 days old (Fig. 2). This

period coincides with the foraging activity

of worker bees. For native polyacrylamide

gel, the ag activity was high from samples

of bees 18 to 29 days old. This data is simi-

lar to the result obtained from enzyme as-

say (Fig. 3).

The ag activity for these experiments

was obtained from crude extract. In the fu-

ture, ag from hpg of A. cerana indica will

be purified. Due to RT-PCR, the cDNA

product in foragers was higher than that in

nurse bees and worker bees younger than 6

days old (Fig. 4). According to the assay of

ag specificity, activity staining on native

polyacrylamide gel, and RT-PCR, it is visi-

ble that ag is partially expressed in nurse

bees and fully expressed in foragers. This

result of expression is the same as in

A. mellifera (O h a s h i et al. 1996 and

1997).

ACKNOWLEDGMENTS

We would like to thank Thailand Re-

search Fund, grants # MRG4780007 and

RTA4580012. Also, we are really thankful

to Prof. dr J. Woyke for all useful sugges-

tions and comments.

REFERENCES

Brouwer E.V.M. (1982)– Measurement ofhypopharyngeal gland activity in the honey-bee. Journal of Apicultural Research, 21(4):193 – 198.

DADE, H.A. (1994)– Anatomy and Dis-section of the Honeybee. The Alden, Press;Oxford, UK.

Huang Z.Y., Robinson G.E., Tobe S.S.,

Yagi K.J., Strambi C., Strambi A.,

Stays B. (1991)– Hormonal regulation ofbehavioural development in the honey bee isbased on changes in the rate of juvenile hor-mone biosynthesis. Journal of Insect Physio-logy, 37(10): 733 – 741.

Huber R. E., Mathison R. D. (1976)–Physical, chemical and enzymatic studies onthe major sucrase of honey bees (Apismellifera). Canadian Journal of Biochemis-try, 54: 153 – 164.

Kubo T., Sasaki M., Nakamura J.,

Sasagawa H., Ohashi K., Takeuchi H.,

Natori S. (1996)– Change in the expres-sion of hypopharyngeal gland proteins of theworker honeybees (Apis mellifera L.) withage and/or role. Journal of Biochemistry,119: 291 – 295.

Vol. 50 No. 2 2006 Journal of Apicultural Science 11

Ohashi K., Sawata M., Takeuchi H.,

Natori S., Kubo T. (1996)– Molecularcloning of cDNA and analysis of expressionof the gene for alpha-glucosidase from thehypopharyngeal gland of the honeybee Apismellifera L. Biochemical and BiophysicalResearch Communications, 221: 380 – 385.

Ohashi K., Natori S., Kubo T. (1997)–Change in the mode of gene expression ofthe hypopharynreal gland cells with anage-dependent role change of the workerhoneybee Apis mellifera L. European Jour-nal of Biochemistry, 249: 797-802.

Sasagawa H., Sasaki M., Okada I.

(1989)– Hormonal control of the division oflabor in adult honeybee (Apis mellifera L.) I.Effect of methoprene on corpora allata andhypopharyngeal gland, and it’s alpha -glucosidase activity. Applied Entomologyand Zoology, 24(1): 66 – 77.

Takewaki S., Chiba S., Kimura A.,

Matsui H., Koike Y. (1980)– Purifica-tion and properties of alpha-glucosidase ofthe honey bee Apis mellifera L. Agriculturaland Biological Chemistry, 44(4): 731 – 740.

Takewaki S., Kimura A., Kubota M.,

Chiba S. (1993)– Substrate specificity andsubsite affinites of honeybee alpha--glucosidase II. Bioscience Biotechnologyand Biochemistry, 57(9): 1508 – 1513.

Tanimura T., Kitamura K., Fukuda T.,

Kikuchi T. (1979) – Purification and par-tial characterization of three forms of al-pha-glucosidase from the fruit fly Drosophilamelanogaster. Journal of Biochemistry, 85:123 – 130.

Yao X., Rebecca M., Byers L. (2003)–Multiple sugar binding sites in alpha--glucosidase. Biochemica et Biophysica Acta,1645: 22 – 29..

EKSPRESJA GENU ALFA-GLUKOZYDAZY W GRUCZO£ACH

GARDZIELOWYCH PSZCZO£Y WSCHODNIEJ

Apis cerana indica

C h a n p e n C h a n c h a o , P r a p a i p i t S r i m a w o n g ,

S i r i w a t W o n g s i r i

S t r e s z c z e n i e

Obowi¹zki pszczó³ robotnic w ulu zmieniaj¹ siê wraz z wiekiem. Ciekawym jest zagadnienie

czasowej i przestrzennej ekspresji zwi¹zanych z tym genów, które podlegaj¹ regulacji

œrodowiskowej. W celu kontroli wieku Apis cerana indica pszczo³y po wygryzieniu by³y

zaznaczane farb¹ i zaliczane do grupy 0-dniowych. Pszczo³y w po¿¹danym wieku pobierano od

0 do 29 dnia w odstêpach trzydniowych. Wyniki wskazuj¹, ¿e gruczo³y gardzielowe (hpg)

zmieniaj¹ swoj¹ wielkoœæ i barwê od du¿ych i kremowych do skurczonych i bladych.

Najwiêksze pêcherzyki stwierdzano u robotnic w wieku od 15 do 18 dni, a najmniejsze

u robotnic 29-dniowych. AktywnoϾ glikozydazy (ag) w surowym ekstrakcie oznaczono jako

nisk¹ u wygryzionych pszczó³. Nastêpnie aktywnoœæ wzrasta³a u robotnic 18 dniowych i by³a

najwy¿sza u robotnic 24-dniowych. Aktywnoœæ surowego ekstraktu oznaczana na natywnym

¿elu poliakrylamidowym, ag u 18 dniowych robotnic by³a niska, a u 29-dniowych wysoka.

Ponadto profil aktywnoœci ag uzyskano przy pomocy RT-PCR. Wskazuje on, ¿e ekspresja ag

u pszczó³ zbieraczek by³a wysoka. Uzyskana sekwencja cDNA wskazuje na œcis³y zwi¹zek

(82,81% podobieñstwa) z cDNA u A. mellifera.

S³owa kluczwe: Apis cerana indica, alfa-glukozydaza, rozwój, ekspresja, karmicielka,

zbieraczka, aktywnoϾ, RT-PCR, cDNA, pochodzenie.

12