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Honey bees in Eastern Arunachal Himalaya: Colony status, Comparison of pH

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Journal of Bioresources 5(2): 58-64 (2018) ISSN 2394-4315

Original Research Article

Honey bees in Eastern Arunachal Himalaya: Colony status, Comparison of pH and Protein
profile (1D SDS) of honey produced

*Hiren Gogoi, Chihi Umbrey, Meth Taba, Mary Badak, Nyabin Riso
Department of Zoology, Rajiv Gandhi University, Rono-Hills, Doimukh-791112, Arunachal Pradesh
Corresponding Author: hirengogoi2007@yahoo.co.in
Received: August 20, 2018; revised: November 20, 2018; accepted: November 25, 2018

Abstract: Honey is a natural sweetener produced by certain Apidae bees. It is widely used for its proven nutritional and
medicinal properties. These properties vary with honey bee species and floral resources they forage. This work was designed
to assess the colony status of the honey bees in eastern part of Arunachal Himalaya, the pH of honey that influence the
antimicrobial and preservative properties and to compare the less known protein profile of the honey in the region. It showed
the presence of 6 species of honey bees viz. Apis andreniformis, A. florea, A. cerana, A. dorsata, A. laboriosa and Trigona
(Lepidotrigona) ventralis var. arcifera with number of colonies in the order 20 (Apis cerana) > 8 (Apis laboriosa) > 4 (Apis
dorsata), 4 (Apis florea) > 1 (Trigona (Lepidotrigona) ventralis var. arcifera). pH of the honey samples was measured as 6.02
(A. cerena), 5.47 (A. florea), 5.45 (A. laboriasa) and 5.16 (A. dorsata). Protein concentration was found to be 58.87  g/mg (A.
dorsata), 45.53  g/mg (A. florea), 43.33  g/mg (A. laboriosa) and 36.67  g/mg (A. cerena). Number of protein bands of
various molecular weights (mw) recorded in different honey samples were 18 ( A. dorsata and A. laboriosa each), 11 (A.
cerena) and 10 (A. florea). All the honey samples resulted 9 similar protein bands with molecular weight ranging between 6.5
- 116.0 kDa. Protein bands of molecular weight between 24.0 - 29.0 kDa, 20.0 kDa, 14.2 kDa were recorded only in the honey
of A. dorsata and A. laboriasa. Variation of the protein bands indicate the varied nutritional and medicinal quality in honey
produced by the honey bee species in the region. Differences in protein concentration and the types of protein with different
molecular weights reflect variation in their foraging resources and processing of honey in the hypo-pharynx. The study also
shows the possibility of sustainable utilization of varied quality of honey produced by the honey bees in the region for
empowering the tribal economy and rural development in the region.
Key words: Arunachal Pradesh, honey bee, honey protein, nesting habitat

Introduction
Honey bees (Hymenoptera: Apidae) are eusocial insects
known from origin of human culture for their unique honey
producing and storing ability to nurse their broods. Since
ancient times, honey is known to be used by human for its
nutritional and therapeutic properties. Besides being consumed
as food, it is used in variety of ways such as in cosmetics and
personal care products due to its skin conditioning and
hydrating properties, as an anti-inflammatory, antioxidant and
58
anti-bacterial agent. Its use also extends in the treatment of
ocular illness, throat infections, constipations, obesity,
rheumatoid arthritis, ulcers, healing of wounds, piles, worm
infestation etc. (Noori et al., 2014).
Honey bees are usually grouped on the basis of their
size and nesting patterns. These are the large sized open
nesting honey bees ( Apis laboriosa Smith, 1871 and Apis
dorsata Fabricius, 1793), medium sized cavity nesting honey
Hiren Gogoi et al., 2018
bees (e.g. Apis cerana Fabricius, 1789, A. mellifera Linnaeus,
1758, A. nigrocincta Smith, 1861, A. nuluensis Tingek, Koeniger
& Koeniger, 1996, A. koschevnikovis Enderlein, 1906), dwarf
open nesting honey bees (Apis florea Fabricius, 1789 and
Apis andreniformis Smith, 1858) and stingless honey bees
(e.g. Trigona (Lepidotrigona) ventralis arcifera Cockerell, 1929,
Melipona spp.) which are cavity nesters. Out of these, Apis
mellifera and Apis cerana are mainly reared commercially
(Otis, 1996; Radloff et al., 2011; Phiancharoen et al., 2011).
Honey as defined by Codex Alimentarius Commission
(2001) is a natural sweet substance produced by certain bees by
transforming the plant nectar they collected, which they store in
their combs. Honey is a concentrated complex aqueous solution
and non-allergic food that consists of mainly sugar, water and
other substances such as proteins, vitamins (vitamin B6, thiamine,
niacin, riboflavin, and pantothenic acid), organic acids, pigments,
minerals (calcium, copper, iron, magnesium, manganese,
phosphorus, potassium, sodium and zinc) and phenolic
compounds (Pontes et al., 2007; Ciulu et al., 2011; Alqarni et al.,
2012). Honey contains 80-85% carbohydrate, 15-17% water,
0.1-0.4% protein, 0.2% ash and minor quantities of enzymes,
amino acids and vitamins.
It is known that the protein content in honey is mainly
enzymes and free amino acids. Composition of proteins in
honey varies with bee species and foraging resources (Lee
et al., 1985; Hermosin et al., 2003; Won et al., 2008). The
proteins and amino acids of honey originate from vegetal
sources and the bee gut during honey synthesis process. Apis
cerana honey is known to contain 0.1-3.3 % protein while in
A. mellifera it is about 0.2%-1.6% (Lee et al., 1998). Though
both animal and vegetal sources are recorded to contribute
the protein and amino acid composition in honey, pollen is
the primary source of honey protein (Bogdanov, 2017).
However, little is known about the protein profile of honey
which has nutritional and preservative importance.
PH of a food is one of the several important factors
that determine the survival and growth of microorganisms during
processing and storage of food. The acidity of foods has been
used for centuries to preserve foods. Therefore, study of the pH
value will help to understand the storability of honey.
Honey bees in eastern Arunachal Himalaya
Global honey production is recorded to be approximately 1.20
million tons per annum (Bogdanov et al., 2008). Countries
like Mexico, Turkey, China, Argentina, Ukraine and United
States are being the major producers (Meo et al., 2017).
However, in spite of huge scope with the diverse vegetation
pattern and presence of different species of honey bees, these
are not exploited in Arunachal Himalaya.
Therefore, present investigation was aimed to study
the population status of honey bees in Eastern part of
Arunachal Himalaya, the pH and comparison of protein profile
of the honey produced in the region.
Materials and methods
Population status of honey bee
Field survey for estimating the colony status of honey bees in
Eastern part of Arunachal Himalaya was conducted in East
Siang, Lower Dibang Valley, Dibang Valley and Anjaw district
of Arunachal Pradesh during the period from November, 2017
to January, 2018. Nesting sites and their ecology were visually
monitored. Species were identified based on the following
characters (Bingham, 1897; Schwarz, 1939; Sakagami et al.,
1980; Marks et al., 2013):
(1) Body black, the basal two abdominal segments more
or less red. Pubescence on head and thorax white. Narrow
transverse bands of silky white pile at the base of 2 nd to 5th
abdominal segment................ Dwarf honey bee Apis florea
Fabricius, 1789.
(2) Body black, narrow transverse bands of silky white
pile at the base of 2nd to 5th abdominal segment ............. Dwarf
honey bee Apis andreniformis, Smith, 1858.
(3) Head, thorax and apical abdominal segment black.
Scutellum and basal five segments testaceous yellow. Area
between costa and subcosta dark. Media of hind wing extends
beyond the radio-medial cross vein............ Indian honey bee
Apis cerana Fabricius, 1789.
(4) Head, thorax, legs and apical three segments of
abdomen black. More or less pale and fuscous on hinder part
of thorax and abdomen. Basal three segments of abdomen
honey yellow.......... Giant honey bee Apis dorsata
Fabricius, 1793.
59
Hiren Gogoi et al., 2018
(5) Abdominal tergites of workers of A. laboriosa are
homogeneously black in colour with a white stripe on each
tergite. Long tawny yellow hairs on tergum 1.......... Himalayan
cliff bee Apis laboriosa Smith, 1871.
(6) Wing hamuli 6 in number. Tessellation on cuticle,
yellow scale. Dark spots on metasomal tergum 1......... Trigona
(Lepidotrigona) ventralis var. arcifera Cockerel, 1929.
Collection of honey samples
Honey samples were collected from different regions of eastern
part of Arunachal Pradesh i.e. Apis florea and Apis dorsata
from Lower Dibang Valley, Apis cerana from East Siang and
Apis laboriosa from Dibang Valley.
P H of honey samples
PH of different honey samples viz. Apis florea, Apis cerana,
Apis dorsata and Apis laboriosa were measured using PH
meter (Eutech pH700).
Isolation and purification of proteins
For extraction of protein, 3.0 mg of honey was collected in 1.5
ml centrifuge tube for each of the honey samples (Apis florea,
Apis cerana, Apis dorsata and Apis laboriosa). Samples were
lysed using 10% trichloroacetic acid (TCA)-acetone containing
2%  -mercaptoethanol (ME). Secondary compounds from
the honey sample were removed by protein precipitation.
Precipitation was done through storing the sample solution
at -20°C for 24 hours in extracting solution i.e. 10% TCA-
acetone (3.0 mg honey: 1.5ml 10 % TCA-acetone). After
precipitation, the extraction solutions were centrifuged
(eppendorf, 5415R) at 5000 rpm (3214g) for 30 minutes at
4ºC to remove the unwanted materials, adding protease
inhibitor phenylmethylsulfonyl fluoride (PMSF). After
centrifugation, the supernatant was discarded and the pellet
was washed three times in 1 ml ice-cold acetone, each wash
was followed by centrifugation again at 10,000rpm for
5 minutes. Vigorous disruption of the pellets with a glass rod
was done between each wash. Supernatant was discarded
and the pellet was dried, keeping the tube inverted on a filter
60
Honey bees in eastern Arunachal Himalaya
paper. Pellets were resuspended in Laemmli buffer and then
heated to 100°C for 3 minutes to prepare sample for gel
electrophoresis. After boiling, the tube was cooled immediately
in ice. The sample was centrifuged again at 10,000rpm (9240g)
for 10 minutes and supernatant was collected and stored in -
20°C until used. Quantitative estimation of the protein in the
sample was done following the method of Bradford (1976).
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE)
Basic procedure for SDS-PAGE was followed to separate the
proteins extracted from honey samples (Laemelli, 1970). Equal
amount (20µl) of the prepared protein samples was loaded in
each well of 3.9% stacking gel [2.04ml ddH2O, 0.375ml 1M
tris-HCl, pH 6.8, 0.5ml 30% acrylamide, 30µl 10% SDS, 30µl
10 % ammonium persulfate and 3µl tetramethyl ethylene
diamine (TEMED)] and were separated using 15% running
gel [1.38ml distilled water, 1.5ml 1.5 M tris-HCl, pH 8.8, 3ml
30% acrylamide, 60µl 10% SDS, 60µl 10% ammonium
persulfate and 2µl tetramethyl ethylene diamine (TEMED)].
Tris-glycine was used as a tray buffer. Electrophoresis was
performed at 30V for 4h using a mini vertical gel electrophoresis
unit (GeNei). After electrophoresis, proteins were stained with
Coomassie Brilliant Blue R-250 (CBB R-250) for overnight
(Weber et al., 1972). Gel was then destained in a mixture of
40% methanol and 10% acetic acid (40ml methanol, 10ml
acetic acid and 50ml distilled H2O) for clear visualization and
detection of protein bands. Gel was photographed using gel
doc system (Bio Rad). Molecular weights of the separated
proteins were compared with a standard molecular weight
marker (Sigma, protein molecular weight marker 6.5-200
kDa).
Results
Population status of honey bee
During the present study, 6 species of honey bees viz. Apis
andreniformis, A. florea, A. cerana, A. dorsata, A. laboriosa
and Trigona (Lepidotrigona) ventralis arcifera (Fig. 1) were
recorded from eastern part of Arunachal Pradesh covering
Hiren Gogoi et al., 2018 Honey bees in eastern Arunachal Himalaya

the East Siang, Lower Dibang Valley, Dibang Valley and Anjaw
district. Out of these species, Apis cerana was recorded from
all the districts, while A. dorsata was recorded from East
Siang, Lower Dibang Valley and Dibang Valley district;
A. laboriosa from Lower Dibang Valley, Dibang Valley and
Anjaw district; A. florea from East Siang and Lower Dibang
Valley; A. andreniformis and Trigona (Lepidotrigona) ventralis
arcifera from Lower Dibang Valley district. Twenty colonies
of A. cerana, 8 colonies of A. laboriosa, 6 colonies of A. dorsata,
4 colonies of A. florea and 1 colony of L. arcifera were recorded
(Table 1). A. andreniformis were recorded from foraging site
only and no nests were recorded. It shows that Apis cerana
was distributed both in lower and higher altitude, A. florea
and L. arcifera in lower altitude, A. dorsata in lower altitude
to moderate altitude and A. laboriosa in higher altitude.
Further, only A. cerana was found to be reared in the studied Fig 1. A- Apis andreniformis, B- Apis florea, C- Apis cerana, D- Apis
area. Rearing was done using movable hive, hollow tree trunk dorsata, E- Apis laboriosa, F- Trigona (Lepidotrigona) ventralis arcifera.
and concrete walled cabinet. All the A. laboriosa colonies were
combless clusters during the winter season. Combless clusters
were found at altitude ranging from 1,880m to 2,600m above
mean sea level and these were recorded near river banks.
A. dorsata colonies were observed on a concrete water supply
tank. A. florea nests were recorded at height of 0.35-4.2m
above ground, while that of A. cerana at height of 0.30-3.5m,
A. laboriosa at height of 0.50m to 50m, A. dorsata at height of
6m and L. arcifera at a height of 5m from the ground. Colony
of the L. arcifera can only be detected due to the presence of
small pipe like entrance (Fig. 2).
P H of honey samples
PH of the honey samples were recorded to be 6.02 ( A. cerena),
5.47 (A. florea), 5.45 (A. laboriasa) and 5.16 (A. dorsata).
Protein content
Concentrations of the protein extracted from honey samples
were recorded to be 58.87µg/mg ( A. dorsata), 45.53µg/mg
(A. florea), 43.33µg/mg ( A. laboriosa) and 36.67µg/mg
(A. cerena).
Fig. 2. A- Colony of Apis florea, B- Concrete cabinet hive for Apis cerana,
SDS-PAGE protein bands
C- Entrance of Apis cerana nest in wild, D- Drum hive for Apis cerana, E-
Number of protein bands of various molecular weights Colony of Apis dorsata, F- Apis laboriosa combless cluster, G and H- Entrance
recorded in the honey samples were 18 ( A. dorsata and of Trigona (Lepidotrigona) ventralis arcifera nest.
61
Hiren Gogoi et al., 2018 Honey bees in eastern Arunachal Himalaya

Table 1. Distribution pattern and nesting habitat of honey bees in Eastern weight of 29.0kDa was recorded only in the honey of A. dorsata
Arunachal Himalaya. and protein bands of molecular weight between 24.0-29.0kDa,
Sl. Name of the No. of colonies Nesting habitat 20.0kDa, 14.2kDa were recorded only in the honey of
No. bee species recorded
1. Apis laboriosa 8 Rock cliff, combless cluster near A. dorsata and A. laboriosa.
rock cliff of rivers and streams.
2. Apis dorsata 4 Top of water supply tank. Discussion
Hunted by honey harvesters.
Status of honey bee population
3. Apis cerana 20 Cavity in tree trunk, abandoned
box furniture, hollow wooden Apis florea used Lantana camara, Boehmeria sp. and Hibiscus
trunk, concrete box, movable bee rosea as support for construction of their nests. It indicates
hive. their generalist behaviour in selecting nesting plant species.
4. Apis florea 4 Nested on Hibiscus rosea ,
Lantana camara, Boehmeria sp.
Their nests were recorded at distance of 20.5-25m apart, which
Near Fagopyrum esculentum and may be due to their uniform territorial distribution pattern.
Brassica camprestis field. These bees nested in proximity of agricultural foraging fields
6. Trigona 1 Tree trunk at approximate height not more than a distance of 28m or inside the crop fields.
(Lepidotrigona) of 4 m from ground.
ventralis arcifera This may be a strategy to minimize energy budget during
foraging activity and to avoid exposure to enemies. All the
nests of A. florea recorded during the present study were
found within height range of 0.35-4.2m. It agrees with the
finding of Wongsiri et al. (1996) that reported it as 0.5 to 15m
above ground level.
A wild colony of A. cerana in Lower Dibang Valley
was known using the same nest for almost 15 years starting
from 2003 as reported by the land owner. However, the colony
sometime abandons the nest for 1-2 months and again returns
back. It shows the reuse of same nest for many years. Among
the honey bees recorded during this study, only Apis cerana
was found to be domesticated. It is reared commercially in
Lower Dibang Valley and East Sinag district.
Though Apis dorsata was found to construct nest on
Fig. 3. Protein bands of honey samples on 1-D SDS-PAGE, a CBB-R250 trees and other human constructions including building, bridges
staining result. (MWM- Molecular weight marker, 1- A. dorsata, 2- A. etc., during the present study it was only recorded from a
cerana, 3 - A. laboriosa and 4 - A. florea. concrete water supply tank.
A. laboriosa each) , 11 ( A. cerena ) and 10 ( A. florea ) During the present study, Apis laboriosa colonies
respectively. Almost 9 similar protein bands with molecular were found at altitude ranging from 1,200 to 3,500m above
weight ranging between 6.5-116.0kDa and one protein band msl recorded in other region (Roubik et al., 1985). All colonies
less than 6.5kDa were recorded in these honey samples. Protein recorded were comb less clusters. Combless cluster during
bands with molecular weight between 97.0-116.0kDa and winter has also been reported in other places (Underwood,
between 29.0-45 kDa were recorded to be darker (thick) in 1990).
all four types of honey samples. One protein band of molecular
62
Hiren Gogoi et al., 2018
Single colony of Lepidotrigona arcifera was recorded
on a tree at a height of 4m from ground level. Probably due
to low population or presence of the small pipe like sign of
entrance at considerable height which is not clearly visible,
only single colony was recorded during this study.
pH value of honey
pH value of honey was recorded to be highest in A. cerena
(6.02) and lowest in A. dorsata (5.16). This difference in pH
value may be due to difference in level of organic acid
composition. Thus, highest acidic nature of A. dorsata with
pH 5.16 may be attributed to the high level of organic acid
content.
Protein profile of honey
Honey samples collected from different nesting site and stored
without additional preservative were recorded with the
presence of protein. This may be due to presence of active
natural protease inhibitor. Protease inhibitors are molecules
that inhibit the activity of proteases. Protease inhibitors can
be in the form of proteins, peptides or small molecules.
Naturally occurring protease inhibitors are usually proteins
or peptides. The protease inhibitor in honey may be derived
from plant via nectar and pollen which are usually synthesized
by plants as defense molecules against pathogens and
phytophages (Ramirez and Montenegro, 2004).
Concentration of protein was recorded to be highest
in A. dorsata (58.87µg/mg) and lowest in A. cerena (36.67µg/
mg). Simillarly protein bands was recored to be highest in A.
dorsata (18 bands). However, A. florea with protein
concentration 45.53µg/mg was recorded with lowest number
of protein bands (10 bands). Therefore, protein concentration
may not be always proportionate with the number of different
protein bands. This may be due to presence of proteins of
similar molecular weight in the A. florea (protein concentration
45.53µg/mg: 10 protein bands) and presence of proteins with
different molecular weight in A. cerena (protein concentration
36.67µg/mg: 11 protein bands). The difference in protein
concentration and different types of protein with different
molecular weights may be attributed to their habitats
(vegetation pattern), floral visit, biomolecular composition of
Honey bees in eastern Arunachal Himalaya
food resources and processing of honey in the gut by different
bee.
Conclusion
From the present study, it can be concluded that eastern
Arunachal Himalaya still harbours suitable nesting sites for
honey bees indicating the possibility of sustainable utilization
to empower the tribal and rural development. Honey of
A. dorsata with highest protein concentration may be
considered preferable over the honey of A. florea, A. laboriosa
and A. cerena in terms of protein concentration. A. dorsata is
also preferable for storage for longer duration with highest
acidic nature (pH 5.16). Further study can be done on protein
profiling and analysis of protein in the honey to find out the
naturally occurring protease inhibitor which can be used for
pharmaceuticals, drug designing and for food preservation.
Acknowledgements
Authors are thankful to Center with Potential for Excellence
in Biodiversity (CPEB-II), Rajiv Gandhi University for
supporting field survey in Dibang Valley and Anjaw district.
Authors also thankful to Department of Zoology, RGU for
providing necessary laboratory facilities.
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