Physiological Entomology (2017), DOI: 10.1111/phen.

12213

Activity of salivary glands in secreting

honey-elaborating enzymes in two subspecies of
honeybee (Apis mellifera L)
A L I A A A . A L - S H E R I F 1, A D E L M . M A Z E E D 2 ,
M O H A M E D A . E W I S 2, E M A D A . N A F E A 1, E L - S E I D E . H A G A G 1
and A H M E D A . K A M E L 1
1
Beekeeping Section, Plant Protection Institute, Agriculture Research Center, Ministry of Agriculture, Giza, Egypt and 2 Faculty of
Agriculture, Department of Entomology, Cairo University, Cairo, Egypt

Abstract. The activity of invertase, glucose oxidase and amylase in the cephalic
(post-cerebral) and thoracic salivary glands is determined in Egyptian and Carniolan
honeybees (Apis mellifera L). For this purpose, three ages of worker bees are selected
for enzyme assays. The results show that the three target enzymes are detected in the two
glands during the three worker ages, except invertase, which cannot be detected in the
cephalic gland of newly emerged bees of both subspecies. In both glands, the secretion
of invertase is highest, followed by amylase and then glucose oxidase. In Carniolan
bees, invertase secretion of the cephalic and thoracic glands increases gradually with
age. In Egyptian bees, invertase increases with age only in the cephalic gland, whereas,
in the thoracic gland, the highest secretion activity is detected in 10–15-day-old bees.
The highest amounts of glucose oxidase and amylase in the cephalic gland are detected
in newly emerged individuals of both Egyptian and Carniolan bees. In the thoracic
gland, however, the highest activity of both enzymes is recorded only in newly emerged
Egyptian bees. The results are discussed in the light of bee management and biological
aspects of the two subspecies.
Key words. amylase, Apis mellifera lamarckii, Apis mellifera carnica, cephalic and
thoracic glands, foragers, glucose oxidase, invertase, newly emerged bees, nurse bees.

Introduction According to Simpson (1960) and Simpson et al. (1968), the

The study of salivary system functions provides important
insights for understanding the diversity of insect life because
the function of the salivary system in insects is closely related
to their lifestyle. The honeybee salivary system consists of
two secretory glands: the cephalic gland, which is located
in the head, and the thoracic gland in the thorax. The two
glands are connected to a common duct that opens in the
mouthparts (Katzav-Gozansky et al., 2001). The thoracic and
cephalic salivary glands produce different types of secretions
and have different functions, even though they have a common
embryonic origin and excretory duct (Poiani & Cruz-Landim,
2010).
Correspondence: Adel M. Mazeed, Cairo University Street, Giza
11613, Egypt. Tel.: +20 23336 1965, +20 23889 4291; e-mail: adel-
mazeed@hotmail.com
cephalic gland produces an oily secretion, whereas the secretion
produced by the thoracic gland is aqueous and contains digestive
enzymes. The secretion produced by the cephalic gland may
help with wax manipulation (Katzav-Gozansky et al., 2001), the
lubrication of the mouthparts (Simpson, 1960) and scent trail
demarcation (Jarau et al., 2004; Schorcopf et al., 2007). The
secretion of the thoracic gland may be used as a wax softener
and for dissolving food (Schönitzer & Seifert, 1990), as well
as in carbohydrate digestion to produce honey (Shinkhede &
Tembhare, 2016).
The transformation of nectar to honey is a gradual process
and begins when the foragers collect nectar and during the
returning flight (Nicolson & Human, 2008). The nectar collected
by worker bees is mixed with secretions from their salivary and
hypopharyngeal glands (Oddo et al., 1999). In the hive, when
the nectar is passed from bee to bee, more secretions are added,
enabling nectar to ripen into honey (Maurizio, 1975). The main

© 2017 The Royal Entomological Society 1

2 A. A. Al-Sherif et al.
enzymes added to the collected nectar are invertase, glucose
oxidase and amylase, which account for the sugar composition
of honey, comprising of approximately 38% fructose, 31%
glucose, and other di- and tri-saccharids (Doner, 1977). The
bees add invertase to convert sucrose to fructose and glucose,
which are the main sugars in honey (White, 1975). In addition
to invertase, the bees also add glucose oxidase, which oxidates
glucose to gluconic acid and hydrogen peroxide, both of which
afford antiseptic activity to the honey (Ohashi et al., 1999).
Amylase breaks down the starch in honey to maltose and
dextrins (Balasubramanyam & Ramesha, 2012).
This process, and consequently the amount of added enzymes,
depends on various factors, such as age, diet, the physiological
stage of the bees, the strength of the colony, temperature and the
abundance of nectar flow (Brouwers, 1982; Fluri et al., 1982;
Huang & Otis, 1989).
In their study of the hypopharengeal gland, Al-Sherif et al.
(2012) demonstrate the secretion of invertase, glucose oxdase
and amylase and report differences in the amounts of the
secreted enzymes between two honeybee races of western
honeybees at different life stages of the worker bees. The three
enzymes are also found in the hypopharyngeal, post-cerebral,
thoracic and mandibular glands of foragers and house bees of
two honeybee species: Apis dorsata (Balasubramanyam, 2014)
and and Apis cerana indica (Balasubramanyam & Ramesha,
2012; Shinkhede & Tembhare, 2016).
In the present study, we conduct a comparative analysis of
the head and thoracic salivary gland secretions of worker honey
bees with respect to the most important enzymatic constituents,
invertase, glucose oxidase and amylase, which are related to
honey repining, aiming to determine the degree to which the
three enzymes can be detected in the two glands, and also to
test the effect of age and the genetic background of bees on the
quantity of the secreted enzymes.
Materials and methods
For sample collection, three colonies of Carniolan (Apis mellif-
era carnica) and Egyptian bees (Apis mellifera lamarckii) were
chosen randomly. The Egyptian and Carniolan colonies were
obtained from a conservative area in ‘Sahel Selem’ in Assuit
governorate and in Dakahlia Governorate (Manzala), respec-
tively, where the two subspecies are preserved purely.
All of the samples were collected and analyzed in the apiary
and laboratory of the Experimental Station of Plant Protection
Research Institute, Department of Apiculture, Ministry of Agri-
culture in Egypt.
In each of the experimental colonies, an empty worker comb
was inserted into the middle of the brood nest for egg-laying
and was observed until brood emergence. Three groups of bees
were collected for comparative analysis: newly emerged bees
(0–5 days old), nurse bees (10–15 days old) and foragers.
The newly emerged bees of both subspecies were obtained by
taking the matured sealed brood combs and placing these in an
incubator at 34 ∘ C and 70% relative humidity. On emergence in
the incubator, worker bees of each race were marked differently
by use of a colour pen for easy recognition and were allowed to
© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213
move freely in a nursery colony to ensure the same microclimate
conditions for both subspecies. The nursery colony was headed
by a naturally mated hybrid queen and was full-sized, occupying
one standard Langstroth hive body.
Every fifth day, the emerged bees were individually
colour-marked with paint dots on the thorax, using differ-
ent colours at each time, and then were introduced into the
nursery colony; thus, bees were defined as being 0–5 days old
on the day of coding and release into the nursery colony. Nurse
bees were collected after 10 days of the bees being introduced
into the nurse colony, and so the bees were defined as being
10–15 days old. Foragers were collected after depositing a small
bee hive with a brood comb in the location of the bee subspecies
colony under consideration. Accordingly, the forager bees were
collected when they returned to the hive (Ohashi et al., 1999).
Both the cephalic (post-cerebral) and thoracic glands were dis-
sected from worker honeybees of all three ages and then stored in
Eppendorf vials at −20 ∘ C. The glands (70 glands/100 μL) were
homogenated in phosphate buffer (40 mm phosphate-buffered
saline, containing 150 mm NaCl, pH 6.7) and then centrifuged
at 4025g for 5 min (Takenaka et al., 1990a).
Invertase activity
Invertase activity was measured in the culture supernatant
using the method of Sumner & Howells (1935). One milliliter of
the cell-free supernatant was mixed with an equal volume of an
aqueous solution of sucrose (10 g L−1 ) as the substrate dissolved
in 20 mm acetate buffer (pH 5.4). The mixture was incubated
at 37 ∘ C for 15 min. Dinitrosalicylic acid reagent (2 mL) was
then added and the reaction mixture was boiled for 5 min. The
absorbance of the cooled reaction mixture was read at 540 nm
against a blank solution using the method of Miller (1959). The
blank was prepared in the exact same way as the sample with the
exception that pure water was added instead of the sample. One
unit of invertase activity was defined as the amount of enzyme
required to liberate 1 μmol glucose mL−1 min –1 at 37 ∘ C.
Glucose oxidase activity
Glucose oxidase activity was assayed by the method of White
et al. (1963). 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−1 o-dianisidine. To determine the amount of H2 O2
liberated from the glucose, 1 μL of 0.04 μg mL−1 peroxidase and
10 μL of test sample were 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. Absorbance at 400 nm (oxidized o-dianisidine) was
then measured using a spectrophotometer. The glucose oxidase
activity of the samples was determined using the standard curve
of H2 O2 , where the glucose oxidase unit (u) is defined as the
amount of enzyme that generates 1 μmol of H2 O2 min –1 at
37 ∘ C.
 Honey-elaborating enzymes in salivary glands 3

Amylase activity Table 1. Split-plot analysis of variance for studying the change of enzyme
activity of the cephalic gland in relation to ages and subspieces of honeybees.
Amylase activity was assayed by a dinitrosalicylic acid pro-
Glucose oxidase Invertase Amylase
cedure (Bernfeld, 1955). The assay was performed in a total
volume of 100 μL of 0.1 m sodium phosphate buffer (pH 6.1) Source of variation d.f. SS F SS F SS F

containing 1% soluble starch as the substrate. After incubation Subspecies (A) 1 0.24 12.78* 1646.04 1141.28* 64.44 129.2*
for 60 min at 37 ∘ C, an equal volume of dinitrosalicylic acid Error A 4 0.07 5.76 1.99
was added to stop the reaction. To determine the amount of Ages (B) 2 2.64 64.63** 29183.9 5883.9** 1162.18 1011.6**
A×B 2 0.6 14.68* 1527.89 308.04** 178.28 155.19**
glucose liberated, the solution was boiled for 5 min and then
Error B 8 0.16 19.83 4.59
diluted 10-fold with water. Absorbance was measured at 550 nm
using a spectrophotometer. The diastase activity of a sample *P < 0.05; **P < 0.01.
was determined using the standard curve of glucose. One unit
of enzyme activity was defined as the amount of enzyme that Table 2. Split-plot analysis of variance for studying the change of enzyme
releases 1 μmol glucose min –1 as reducing sugar under the assay activity of the thoracic gland in relation to ages and subspieces of honeybees.
conditions.
Glucose oxidase Invertase Amylase

Source of variation d.f. SS F SS F SS F

Statistical analysis
Subspecies (A) 1 10.98 54.79* 181.32* 270.76* 349.36 290.66*
Error A 4 0.80 2.67 4.80
First, data were adjusted by logarithmic transformation to Ages (B) 2 85.23 236.8** 9364.99 1326.7** 1229.4 452.45**
improve normality of the distribution and to equalize vari- A×B 2 29.28 81.35** 12721.47 1802.2** 310.02 114.09**
ance between groups and also analyzed as such. Because of Error B 8 1.43 28.23 10.86
the similarity of the results before and after transformation, *P < 0.05; **P < 0.01.
the data were analyzed using the original values. Because
age factor was nested within each colony, the data con-
formed to a split-plot design and were analyzed as such.
The subspecies were treated as the main units and ages as
subunits. In addition, the simple main effect of subspieces
and age was tested in accordance with Clewer & Scarisbrick
(2001). The data were analyzed using the Almo-Statistic-System
(Holm, 2013).

Results

The results of statistical analysis of the data are presented in

Tables 1 and 2. The subspecies and the age of worker bees, as
well as the interaction between them, are seen to have significant Fig. 1. Change of invertase activity in cephalic gland of Carniolan
influences on enzyme activity in both glands. (Car) and Egyptian (Lam) bees as workers age (vertical lines indicate
the SD).

Effect of subspecies (main plot effect) Invertase

The difference between the two subspecies, averaged over the
three life stages of bees, was significant. However, there may
be interaction (i.e. the difference between the two subspecies
may not maintain a consistent difference for the three ages under
study) (Figs 1–6 and Tables 1 and 2).
Effect of age (subplot) and the interaction between age
and subspecies
The results suggest a significant effect of age on the
activity of the three enzymes under study but, because
of the large age × subspecies interaction value, the ages
should be considered for each subspecies (Figs 1–6 and
Tables 3 and 4).
In the cephalic gland, invertase activity was absent in the two
subspecies in newly emerged bees but, at 10–15 days, the gland
began to secrete invertase in both subspecies and it reached its
maximum value in forager bees.
The two subspecies differentiated significantly from each
other with respect to both nurse bees and foragers, being higher
in Egyptian bees than Carniolan bees [least significant difference
(LSD): P < 0.05].
Concerning the thoracic gland in newly emerged bees,
there was no enzyme activity in Carniolan bees, although a
small amount of secretion was detected in Egyptian bees. At
10–15 days, the secretion began to increase significantly in both
subspecies, although it exhibited a different trend in foragers,
being decreased dramatically and significantly in Egyptian bees
and increased in Carniolan bees (LSD: P < 0.05).

© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213

4 A. A. Al-Sherif et al.

Fig. 5. Change of amylase activity in cephalic gland of Carniolan (Car)

Fig. 2. Change of invertase activity in thoracic gland in Carniolan (Car)
and Egyptian (Lam) bees as workers age (vertical lines indicate the SD).
and Egyptian (Lam) bee as workers ages (vertical lines indicate the SD).

Fig. 6. Change of amylase activity in thoracic gland of Carniolan (Car)

Fig. 3. Change of glucose oxidase activity in cephalic gland of
and Egyptian (Lam) bees as workers age (vertical lines indicate the SD).
Carniolan (Car) and Egyptian (Lam) bees as workers age (vertical lines
indicate the SD).
Table 3. Pairwise comparison between Carniolan and Egyptian bees at
various stages of worker bee life.

Invertase Glucose oxidase Amylase

Glands Ages C versus L C versus L C versus L

Cephalic Newly emerged 0 −0.06* 0.27*

Nurse −0.4* −0.11* 0.08*
Foragers −0.21* 0.05* −0.19*
Thoracic Newly emerged −0.66* −0.01 −0.38*
Nurse −0.58* −0.03 0.04
Foragers 0.78* 0.29* −0.17*

Least significant difference: *P < 0.05.

Fig. 4. Change of glucose oxidase activity in thoracic gland of
Carniolan (Car) and Egyptian (Lam) bees as workers age (vertical lines
indicate the SD).
The two subspecies differed significantly from each other at
the three ages, being higher in both newly emerged and nurse
bees in favor of Egyptian bees, but at the forager stage, the
Carniolan bees exceeded the Egyptian bees (LSD: P < 0.05).
Glucose oxidase
In the cephalic gland, the two subspecies exhibited a similar
trend of enzyme activity for the first two life stages of bees, being
decreased significantly from newly emerged to nurse bees (LSD:
C, Carniolan bees; L, Egyptian bees.
P < 0.05). At the forager stage, however, it began to increase
significantly in Carniolan bees (LSD: P < 0.05) and decrease
slightly and nonsignificantly in Egyptian bees (LSD: P > 0.05).
In both newly emerged and nurse bees, the Egyptian bees
exceeded the Carniolan bees with respect to enzyme activity,
whereas, at the forager stage, the Carniolan bees significantly
exceeded the Egyptian bees (LSD: P < 0.05).
In the thoracic gland, the two subspecies maintained the same
trend of enzyme activity for the three life stages of worker bees.
Secretion was high in newly emerged and forager bees but low in
nurse bees. The two subspecies differed, however, significantly
from each other only at the foraging stage, being higher in
Carniolan bees than in Egyptian bees (LSD: P < 0.05). For each

© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213

 Honey-elaborating enzymes in salivary glands 5

Table 4. Pairwise comparison between different life stages of workers in oxidase and invertase are secreted from the hypopharengeal
each subspieces. gland. Furthermore, Ohashi et al. (1999) detected both glucose
oxidase and amylase in the hypopharengeal gland.
Invertase Glucose oxidase Amylase
Al-Sherif et al. (2012) reported that the hypopharengeal gland
Glands Ages C L C L C L produces invertase, glucose oxidase and amylase. In comparison
Cephalic N versus S −0.94* −1.34* 0.18* 0.12* 0.88* 0.68* with the present study, we note that invertase is secreted in
N versus F −1.85* −2.06* 0.05* 0.16* 0.93* 0.46* even higher quantities in the cephalic and thoracic glands than
S versus F −0.91* −0.72* −0.12* 0.04 0.06* −0.22* in the hypopharengeal gland in both Egyptian and Carniolan
Thoracic N versus S −1.08* −1.01* 0.38* 0.36* 0.51* 0.93*
bees. However, the trend for secreting invertase in the cephalic
N versus F −1.53* −0.09 −0.18* 0.12* −0.02 0.19*
S versus F −0.44* 0.91* −0.56* −0.24* −0.52* −0.73* gland of the two subspecies is similar to that found in the
hypopharengeal gland. In both glands, the secretion increases
Least significant difference: *P < 0.05.
as the bees age. In the thoracic gland, however, the synthesis
C, Carniolan bees; L, Egyptian bees; N, newly emerged bees; S, nurse bees; F, foragers.
of invertase reaches its highest peak earlier in Egyptian bees (at
10–15 days) than in Carniolan bees.
subspecies, there were significant differences in enzyme activity Having returned to their colonies, forager bees pass the nectar
between the three ages (LSD: P < 0.05). to special ‘receiver’ bees, which are middle-aged bees that have
not started foraging. The receiver bees add more enzymes to
the nectar before depositing it into comb cells. Accordingly,
Amylase in addition to secreting a high amount of invertase from the
cephalic gland at the foraging stage, the Egyptian bees also add
Concerning amylase activity in the cephalic gland, the highest high quantity of it from the thoracic gland at the nurse stage.
enzyme activity in both subspecies was observed in newly In Carniolan bees, however, only the cephalic gland secretes a
emerged bees, and then it began to decrease significantly high quantity of invertase at the foraging stage. Accordingly, in
and reached approximately the same level at 10–15 days for Egyptian bees, higher quantities of invertase was added to nectar
both subspecies (LSD: P < 0.05). Concerning foragers, the at two life stages that could be reflected in the quality of the
enzyme activities in the two subspecies began to differentiate honey produced because adding a greater quantity of enzymes to
significantly from each other, being increased in Egyptian bees nectar has a positive effect on the quality of the resulting honey
and decreased in Carniolan bees (LSD: P < 0.05). (Albanese, 1952).
The difference between the two subspecies was significant for The high amount of enzyme secretion at an earlier stage in
the three ages, being higher in Carniolan bees than in Egyptian the life of Egyptian bees could be attributed to the fact that
bees for the first two ages, but at the foraging age, the quantity the life span of Egyptian bees is shorter than that of Carniolan
of the secreted enzyme in the Egyptian bees exceeded that of bees (El-Banby, 1956), such that the different life stages of the
Carniolan bees (LSD: P < 0.05). Egyptian bees may begin earlier than in the Carniolan bees.
In the thoracic gland, although the two subspecies exhibited Consequently, it can be expected that the forager stage, which is
similar enzyme activity for the three life stages of bees, the responsible for nectar collecting, may begin earlier in Egyptian
trend was sharper in Egyptian than in Carniolan subspecies. bees than in Carniolan bees.
Although it began to decrease significantly from newly emerged Many beekeepers aim to activate their colonies early in the
to nurse bees, it began to increase significantly in foragers (LSD: season by feeding them with sugar syrup prior to the nectar flow
P < 0.05). There were significant differences between the two so that they can produce a high number of forager bees at the
subspecies in favor of Egyptian bees in both newly emerged and beginning of the flowering season. The foragers are assumed
forager bees (LSD: P < 0.05). to be responsible for producing enzymes that are important
with respect to converting nectar to honey (Simpson et al.,
1968; Takenaka et al., 1990a; Ohashi et al., 1999; Costa &
Discussion Cruz-Landim, 2002). In the present study, invertase is produced
in higher quantities at the forager stage than at the other life
The results of the present study show that the three enzymes, stages in Carniolan bees but, in Egyptian bees, it could be
invertase, glucose oxidase and amylase, were detected in the also produced in high quantities early at the nurse stage. The
two glands in bees of all ages in the two subspecies, except nurse stage in the present study was presumably determined to
for invertase, which was not detected in the cephalic glands comprise bees that were aged between 10 and 15 days. This age
of newly emerged Carniolan and Egyptian bees. Kratky (1931) may coincide with the foraging age of Egyptian bees because,
and Kosmin & Komarov (1932) did not find invertase and as noted above, the foraging age of Egyptian bees may be earlier
amylase in the labial glands but, instead, it was present in than that of Carniolan bees. So, in our situation, the Carniolan
the hypopharengeal gland. Inglesent (1940) detected invertase bee colonies may have to be fed earlier than the Egyptian bees
activity in the thoracic gland but not in the hypopharengeal or before the beginning of the season.
cephalic glands. By contrast, Simpson (1960) and Arnold & By contrast and concerning amylase, the results show that the
Delage-Darchen (1978) noted that the hypopharengeal gland is highest quantity of this enzyme is secreted by newly emerged
the only gland in the salivary system of A. mellifera that has bees in the cephalic and thoracic glands. The trend for secreting
invertase activity. Takenaka et al. (1990b) reported that glucose amylase in the thoracic gland in the two subspecies is similar

© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213

6 A. A. Al-Sherif et al.
to that found in the hypopharengeal gland, as indicated by the
results of a previous study (Al-Sherif et al., 2012).
The growth of newly emerged bees starts as soon as they
begin to feed on pollen, with a resulting increase in longevity,
development of brood food glands, fat bodies and other organs
(Maurizio, 1954). The bees need amylase to catalyze starch
in the pollen or honey to monosaccharides (Crane, 1990;
Balasubramanyam & Ramesha, 2012). Workers can utilize
starch by converting it to glucose during flight, whereas drones
cannot. This provides evidence that amylase found in the
salivary glands of older worker bees or foragers is important
for increasing their efficiency with respect to utilizing complex
carbohydrates and also emphasizes the important role of these
glands as food processors in the highly evolved social system of
the honeybee society (Hrassnigg et al., 2003).
The results of the present study may be important for two
reasons. The difference in enzyme activity between the two
subspecies (i) could be reflected in the chemical composition
of the resulting honey and (ii) also encourage us to establish
a selective breeding programme for selecting bees with a high
quantity of enzyme production.
Acknowledgement
The authors declare that they have no conflicts of interest.
References
Albanese, A.A. (1952) Utilization and protein-sparing action of fructose
in man. Metabolism, 1, 20–25.
Al-Sherif, A.A., Mazeed, A.M. & Hagag, E.E. (2012) Activity of
hypopharengeal gland in secreting honey-elaborating enzymes in
Carniolan and Egyptian honeybees. Egyptian Academic Journal of
Biological Science, 5, 167–173.
Arnold, G. & Delage-Darchen, B. (1978) Nouvelles donn’ees sur
l’ ́ equipment enzimatique des glandes salivaries de l’ouvrier
d’Apis mellifera (Hymenoptera Apidae). Annales des Sciences
Naturelles – Zoologie et Biologie Animale, 20, 401–422.
Balasubramanyam, M.V. (2014) Role of different enzymes in nectar to
honey transformations in indigenous rockbee, Apis dorsata. Journal
of Chemical, Biological and Physical Sciences, 4, 361–368.
Balasubramanyam, M.V. & Ramesha, I. (2012) Amylase and starch
content in ripening of honey of indigenous hive bee Apis cerana
indica. Journal of Chemical, Biological and Physical Science, 2,
237–241.
Bernfeld, P. (1955) Amylases, α and β. Methods in Enzymology, 1,
149–158.
Brouwers, E.V.M. (1982) Measurement of hypopharyngeal gland activ-
ity in the honeybee. Journal of Apicultural Research, 21, 193–198.
Clewer, A.G. & Scarisbrick, D.H. (2001) Practical Statistics and
Experimental Design for Plant and Crop Science, 1st edn. John Wiley
& Son, U.K.
Costa, R.A. & Cruz-Landim, C. (2002) Enzymatic activity of hypopha-
ryngeal gland extracts from workers of Apis mellifera (Hymenoptera,
Apidae, Apinae). Sociobiology, 40, 403–411.
Crane, E. (1990) Bees and Beekeeping – Science, Practice and World
Resources. Comstock Publishing Associates, Ithaca, New York.
Doner, L.W. (1977) The sugars of honey – a review. Journal of the
Science of Food and Agriculture, 28, 443–456.
© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213
El-Banby, M. (1956) The biology and behavior of the Egyptian honey
bee (Apis mellifera lamarckii). M.Sc, Faculty of Agriculture, Ain
Shams University.
Fluri, P., Lüscher, M.M., Wille, H. & Gerig, L. (1982) Changes in
weight of the pharyngeal gland and haemolymph titres of juvenile
hormone, protein and vitellogenin in worker honey bees. Journal of
Insect Physiology, 28, 61–68.
Hrassnigg, N., Brodschneider, R., Fleischmann, P. & Crailsheim, K.
(2003) Worker bees (Apis mellifera L.) are able to utilize starch as fuel
for flight while drones are not. 38th Beekeeping Congress, Apimondia,
24 -29 August, 2003, Ljubljana, Slovenia.
Holm, K. (2013) Almo-Statistik-System. University of Linz, Austria.
Huang, Z.Y. & Otis, G.W. (1989) Factors determining hypopharyngeal
gland activity of worker honey bees (Apis mellifera L.). Insect
Sociaux, 36, 264–276.
Inglesent, H. (1940) Zymotic function of the pharyngeal, thoracic and
post-cerebral glands of Apis mellifera. Journal of Biochemistry, 34,
1415–1418.
Jarau, S., Hancir, M., Zucchi, R. & Barth, F.G. (2004) A stingless bee
uses labial gland secretions for scent trail communication (Trigona
recursa S.). Journal of Comparative Physiology: (A Neuroethology,
Sensory, Neural and Behavioral Physiology), 190, 233–239.
Katzav-Gozansky, T., Soroker, V., Ionescu, A. et al. (2001) Task-related
chemical analysis of labial gland volatile secretion in worker hon-
eybees (Apis mellifera ligustica). Journal of Chemical Ecology, 27,
919–926.
Kosmin, N.P. & Komarov, P.M. (1932) Ueber das
Invertierungsvermogen der Speicheldruesen und des Mitteldarms
von Bienen verschiedenen Alters. Zeitschrift fuer Vergleichende
Physiologie, 17, 267–279.
Kratky, E. (1931) Morphologie und Physiologie der Druesen in Kopf
und Thorax der Honigbiene (Apis mellifica L.). Zeitschrift fuer
Wissenschaftliche Zoologie, 139, 120–200.
Maurizio, A (1954) Pollenernährung und Lebensvorgänge bei der Hon-
gibiene (Apis mellifica L). Landwirtschaftliches Jahrbuch Schweiz,
62, 115–182.
Maurizio, A. (1975) How bees make honey. Honey: A Comprehensive
Survey (ed. by E. Crane), pp. 77–105. Heinemann, U.K.
Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for the determi-
nation of reducing sugar. Analytical Chemistry, 31, 426–428.
Nicolson, S.W. & Human, H. (2008) Bees get a head start on honey
production. Biological Letters, 4, 299–301.
Oddo, L.P., Piazza, M.G. & Pulcini, P. (1999) Invertase activity in honey.
Apidologie, 30, 57–65.
Ohashi, K., Natori, S. & Kubo, T. (1999) Expression of amylase and
glucose oxidase in the hypopharyngeal gland with an age-dependent
role change of the worker honeybee (Apis mellifera L.). European
Journal of Biochemistry, 265, 127–133.
Poiani, S.B. & Cruz-Landim, C. (2010) Changes in the size of
cephalic salivary glands of Apis mellifera and Scaptotrigona postica
(Hymenoptera: Apidae) queens and workers in different life phases.
Zoologia, 27, 961–964.
Schönitzer, K. & Seifert, P. (1990) Anatomy and ultrastructure of the
salivary gland in the thorax of the honeybee worker, Apis mellifera
(Insecta, Hymenoptera). Zoomorphology, 109, 211–222.
Schorcopf, D.L.P., Jarau, S., Francke, W. et al. (2007) Spitting out
information: Trigona bees deposit saliva to signal resource locations.
Proceedings of the Royal Society of London Series B: Biological
Sciences, 274, 895–898.
Shinkhede, M.M. & Tembhare, D.B. (2016) Digestive enzyme secretion
by thoracic salivary glands in honey bee Apis cerana indica (Hymen-
opera: Apidae). Indian Journal of Entomology, 78, 223–228.
Simpson, J. (1960) The functions of the salivary glands of Apis mellifera.
Journal of Insect Physiology, 4, 107–121.

Simpson, J., Riedel, I.B. & Wilding, N. (1968) Invertase in the hypopha-
ryngeal glands of the honeybee. Journal of Apicultural Research, 7,
29–36.
Sumner, J.B. & Howells, S.F. (1935) A method for determination of
saccharase activity. Journal of Biological Chemistry, 108, 51–54.
Takenaka, T., Ito, H., Yatsunami, K. & Echigo, T. (1990a) Changes
of glucose oxidase activity and amount of gluconic acid formation
in the hypopharyngeal glands during the lifespan of honey bee
workers (Apis mellifera L.). Agricultural and Biological Chemistry,
54, 2133–2134.
Takenaka, T., Miwa, S. & Echigo, T. (1990b) Changes of protein content
and enzyme activity in hypopharyngeal glands during lifespan of
© 2017 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12213
Honey-elaborating enzymes in salivary glands 7
honeybee workers (Apis mellifera L.). Bulletin of the Faculty of
Agriculture, Tamagawa University, 30, 1–8.
White, J.W. (1975) Composition of honey. Honey: A Comprehensive
Survey (ed. by E. Crane), pp. 180–194. Heinemann, U.K.
White, J.W., Subers, M.H. & Schepartz, A.I. (1963) The identification of
inhibine, antibacterial factor in honey, as hydrogen peroxide, and its
origin in a honey glucose oxidase system. Biochimica et Biophysica
Acta, 73, 57–70.
Accepted 12 July 2017