Chapter I

Introduction and Review of Literature

The honeybee and the fruit of it~ toils have been familiar to us from

prehistoric times (Singh, 1962). Mesolithic rock paintings of honey collection at

Ganeshghati and Bhonrawali hill, Central India (Mathpal, 1984) reveal human

interest for honey. This winged creature finds mention in the Vedas and

Ramayana (existence of Madhuban), the Book of Proverbs, the Quran and

many other ancient books (Singh, 1962). It is one of the few social insects that

are directly beneficial to man. The honeybee belongs to order Hymenoptera

(Family Apiidae) under phylum Arthropoda and class Insecta. Among more

than 3,00,000 species of Hymenoptera, the honeybee is one of the most

important insects. One of the reasons, from man's point of view, is production

of honey and value-added products like wax, royal jelly, pollen, propolis and

venom. Honeybees also have a role in pollination. The honeybee is one of the

most well studied social insects and there is enormous information available

mainly in the field of apiculture. However, most of the work has been done on

Apis mellifera, the European honeybee. Very little information is available on

the behaviour and ecology of other species in the genus Apis (Sakagami, 1960).

We have focused our study on the giant Asian honeybee i.e., Apis dorsata that

is not a very popular study object because of its ferocious temperament. These ..

bees are easily provoked and when enraged they pursue their victim over long

1

distances (Singh, 1962). They are considered as one of the most ferocious

stinging insects on earth (Morse and Laigo, 1969). These bees (bigger than

most of the honeybee sp.) nest in the open. Generally the hive is of single layer

and huge in size (5-6 ft x 3 ft) (Butler, 1954). As the hive is easily visible and

quite attractive to other animals for large collection of honey and brood, Apis

dorsata prefers to make hives in tall trees and historical buildings to protect

their hive from easy access to other animals. That is another reason why it is

difficult to study the behaviour of Apis dorsata. Considerable efforts have been

made in the past to domesticate these bees as they can produce large amount of

honey, but nobody has succeeded till date.

The present study deals with the different behavioural aspects of Apis

dorsata. Natural Apis dorsata hives outside the building of School of Life

Sciences, Jawaharlal Nehru University, New Delhi (Longitude 77.12E and

latitude 28.35N), hanging from the third floor sunshade, were used for these

studies. They were about 3-4 feet away from the glass window of our

laboratory. Most of the hives faced (as they had 2 sides - one of them faced the

sun and the other side faced the opposite direction) the easterly direction. The

. fact that these bees did not get disturbed on opening the window seemed to

2

show that they were conditioned to this activity, making it possible to observe

them keeping the natural environment undisturbed.

1.1. About Apis dorsata:

1.1.1. Distribution of Apis dorsata:

Apis dorsata occurs abundantly in the wild state both in forests and in

cultivated as well as uncultivated areas of India, Ceylon and other parts of

South Asia (Butler, 1954). In India, this bee is found up to a height of 1,220 m

above sea level (Singh, 1962). Fig. 1.1 shows the geographic distribution of A.

dorsata throughout the world.

1.1.2. The nest and nest site of the giant Asian honeybee:

The comb of Apis dorsata is always attached to the underside of

overhanging rocks; suspended from the more or less horizontal branches of tall

trees such as Banyan (Ficus bengalensis), Pipal (Ficus religiosa), Mango

(Mangifera indica), Jamun (Syzygium cumini) etc. (Verma, 1990), or from the

eaves of tall buildings (Butler, 1954 ). Deodikar et a/. ( 1977) reported 45 per

cent of colonies on terrestrial supports while about 55 per cent were arboreal.

Dozens of colonies may be found on one tree.

3

dorsa to

~ig. 1.1. Global distribution of Apis dorsata with subspecies binghami and brevi/igu/a and yet not definitely classified laboriosa (Sakagami et a/., 1980)

A hive is more or less semi..,circular. The size of a single comb of A.

dorsata, depending upon the season and stage of development of a colony,

measures 1.5-2 m from side to side and 0.6-1.2 m from top to bottom (Deodikar

et al., 1977). A single comb may be as much as 5-6 feet long and 3 feet deep

(Butler, 1954 ). A comb is made up of 2 hexagonal wax cell layers fixed back to

back. It has a definite zone for storage of honey and brood. The upper portions

of the comb store honey and pollen and is generally 10-25 em thick. Below this

storage area is the brood nest (Singh, 1962). The colonies are perennial and the

development of new colonies takes place by swarming (Butler, 1954 ).

1.1.3. Functional organisation of the nest:

The bees in the nest perform various functions based on the hierarchy of

the bees. Some bees (clinging as a thin layer next to the wax cells) construct the

comb, take care of the brood and process honey. Most of the bees in a colony

(about 80-90%) make a thick multi-layered cover called protective curtain

(Morse and Laigo, 1969). There is an air space between the two protective

layers of the nest, which helps to regulate the temperature of the nest. Bees of

the protective curtain remain motionless with their wings spread out. In the

lower part of the hive facing the sun, there is an active zone (Fig.1.2) called the

4

Fig. 1.2. Apis dorsata hive showing a distinct mouth zone.

)f the colony (Morse and Laigo, 1969) whe~e foragers dance, land or

P. • ,oragmg.

'he adult occupants of the nest are thousands of worker bees, a single

1d several hundred drones. The worker bee is light brown in colour. The

darker in colour than the workers are and broader by about 2 mm in the

region (Singh, 1962). The drone is black in colour and has a blunt

r1 without a sting. The drones are not in the hive in the winter

·ick, 1992).

f orker bees are sterile females, arising from fertile eggs (eggs hatch

days), yet are sexually immature because of glandular changes induced

ng with a modified diet after their second day as a larva (Brown, 1988).

~es follow a strict division of labour according to their age; starting

1 cleaning to foraging.

5

Duties of worker

Cell cleaning; keeping brood area warm

Feeding larvae; attending queen

Fanning and ventilation control (few)

Polishing cells; packing pollen

Orientation flights, around midday

Guard duties; clearing out debris

Collecting pollen (some bees)

Age of

worker (days)

0-4

3-14

3-22

4-24

5-18

10-18

10-26

Collecting nector 12-35

( from Honeybees: A guide to management by Ron Brown ( 1988); The

Crowood Press)

1.1.4. Apis dorsata as a benificial insect:

1.1.4.1. Production of honey and beeswax:

In India, A. dorsata accounted for about 70% of the honey in 1951 (Ghatge,

1951) and for 80% of the bees wax production in 1961 (Phadke, 1961). A colony may

yield 40-70 kg of honey (Dutta et a!., 1983). Generally the average honey yield per

colony is about 5-10 kg. Singh ( 1962) reported that a single colony might yield up to

37.3 kg honey during a year. Many tribal people in India depend either wholly or

partially on collection of honey and beeswax for their livelihood.

6

1.1.4.2. An efficient pollinator:

Hundreds of species of agricultural plants in over 40 plant families

worldwide are pollinated, at least in part, by bees and other insects (Crane and

Walker, 1984; Free, 1970; Me Gregor, 1976; Southwick and Southwick,

1989b ). Many agricultural crop plants require out-crossing to produce viable

fruit and others show hybrid vigour with the production of a better crop (Mortiz

and Southwick, 1992). The yields of many fruit, vegetable, seed and nut crops

would drop substantially without pollination by honeybees (Morse, 1988;

Olmstead and Wooten, 1987). It has been estimated that honeybees alone

account for as much as 80% or more of all crop insect pollination (Camazine

and Morse, 1988). Apis dorsata is listed as an important pollinator of plants in

several reports (Maun and Singh, 1983 ). Pollination efficiency is related to

flight range and among the honeybees, A. dorsata has the longest flight range

(8,500 m) (Singh, 1962). Because of migratory activity, the foraging range of A.

dorsata is large. A. dorsata is specialized in exploring rich nectar sources even

at distances further than 5 km. (Koeniger and Vorwohl, 1979). These bees can

adapt to extreme climatic conditions. They have been observed (Singh, 1962) to

begin the days work earlier and stop it later than A. indica.

7

1.1.4.3. To treat diseases:

The dreaded bee-sting has the mysterious quality of healing muscular

and nervous pains and aches of sciatica, rheumatism and arthritis (Singh, 1962).

Bee venom therapy for multiple sclerosis has been reported by Mraz (1993).

However, the use of A. dorsata for the above purpose has not been reported as

yet.

1.1.4.4. As a weapon of war:

Bees have been used as a weapon of war through the ages. In World War

I, infuriated swarms were used to hamper the advance of forces in Belgium. In

our country, many a political meeting has ended (Singh, 1962) in pandemonium

after a stone has been thrown by a mischievous opponent in a colony of wild

bees hanging from a branch of a nearby tree.

1.2. Biological aspects related to the vision of honeybee:

The present study is restricted to some specific biological aspects related

to vision of Apis dorsata. The following section discusses these in more detail

keeping the literature in view.

8

Vision together with smell, hearing and other senses serves for

recognition of objects and for orientation. Insects are actually able to see an

object and visually find their way towards it or away from it. Light is the factor

which allows this discrimination and recognition of objects, since it transmits

information about them. The visual recognition of objects and the

corresponding behavioural reaction of the organism are realized through

various visual stimuli. The information concerning the surroundings is carried

by the light and reaches the insects through their eyes. Such information

concerns colour (i.e., spectral composition), brightness, volume, size (i.e.,

distribution of light in space), polarization, and motion (flashing) (Mazokhin­

Porshnyakov, 1969). Foraging and defensive behaviour are two important

biological aspects of the honeybee which use vision as primary stimuli. From

the neuroethological point of view, foraging is a multiple complex act, which

involves the receipt, interpretation and communication of complex information,

memorization, orientation, flight, food collection and distribution. Defensive

behaviour is an instinctive behaviour, which includes aggression i.e., display

and attack.

Foraging is generally a diurnal activity but in Apis dorsata, night time

foraging is also reported (Dyer, 1985). Besides olfaction, vision is an

9

important part of foraging. The visual organ of honeybee comprises two

compound eyes and three simple eyes (in the centre of the head). These simple

eyes are called ocelli (sometimes also referred to as dorsal ocelli). They look

like small inconspicuous black beads. There are 2 lateral ocelli and 1 median

ocellus. Figures 3a-c show the organisation ofvisua1 parts of the honeybee with

the ocelli and the compound eyes.

Colours are very important for the realization of basic behavioural

reactions such as the choice of food, of a sexual partner, or of a place to refuge.

Of course other aspects of light also play a very important role during the

preliminary choice and initial orientation. The final choice is made with the aid

of smell when the insect is eventually in contact with the object of interest

(Mazokhin-Porshnyakov, 1969). All animals can distinguish objects through the

intensity of the reflected light. But the spectral composition (i.e., the colour) of

the reflected light plays a role in object discrimination only for animals which

possess colour vision. Insects do have colour vision, ~md this has been known

since 1914 (Frisch K von). A lot of work has been done on colour vision of

honeybees (Frisch, 1967; Wells et al., 1981; Harrington et al., 1983). The

colour spectrum of the human eye and the eye of the bee is shown in Fig. 1. 4.

An interesting aspect of bee vision as compared to human vision is that in these

10

c

--=~-:;;~·

Fig. 1.3a. Dorsai view-ofthe,.,he~d sh~wi"~~ the- p~sition ~f ocelli and compound eye(n (visual apparatus) of Apis dorsata.

Fig. 1.3b. Transverse section of the head of an adult bee showing the positions of the

ocellar system and the compound eye.

·Fig. 1.3c. A transverse section of the median ocellus of the honeybee.

Ln is ~biconvex lense, Ct is a thickening of

the head cuticle, dark pigment (Pg) formed in the retini (Ret), a is interstitial cells. Nv is nerve fiber.

Man

Blue· {UI~rovooletl Violet Blue green Green Yellow Orange Red

Invisible , }))--___ 400 --l.S0-500 -550-600- 650-?OJ---- 800 m~ L I I I I I I Invisible Ultraviolet Violet Blue Blue· Green Yellow Orange {Red)

~--.---J green Similar

(Blue region l

Bee

Similar

{Yellow region)

Fig. 1.4. The colours of the spectrum: (above) for the human eye; (below) for the eye of the honeybee .

insects the sensitivity range is markedly shifted toward the shortwave end of the

spectrum.

The compound eye of the honeybee gives a very wide-ranging vision

(Kelsey, 194 7). It has a major role in colour detection. Bees can detect colours

(Frisch, 1967) and stick to a particular colour flower patch (Wells eta!., 1981 ).

Honeybees except A. dorsata have been domesticated as they are one of

the most benificial insects for man. But all the efforts so far to domesticate the

wild honeybees (A. dorsata) have failed (Verma, 1992a). It is only possible to

train them for some specific purpose. A. dorsata has been trained previously

using scented sucrose solution (100~1/ l clove oil, 1.5 M sucrose solution)

(Rathore and Wells, 1995). Training utilizes the learning capability (visual as

well as olfactory) ofbees (Frisch, 1914). In a typical training experiment bees

associate odor or colour as a marker of reward (eg, sucrose solution) (Gould et

al., 1988). It is seen that the basic phenomenology of associative learning is

remarkably similar in vertibtates and invertibrates (Gould and Towne, 1988).

Among invertibrates, it is particularly true for molluscs (Sahley, 1984; Sahley

et a!., 1984) and bees (Menzel, 1983; Bitterman et al., 1983; Menzel and

Bitterman, 1983; Abramson, 1986). The temporal phases of memory appears to

be similar in virtually all animals, including bees (Menzel, 1983, 1984). An

11

early phase of memory, also called short term memory, lasts for minutes to

hours (Pinsker et al., 1970; Carew et al., 1971) and can be easily disrupted or

erased by electroconvulsive shock or by any experience which conflicts with

the information just learned. In bees, the stored association of one colour with

could be erased by the presentation of a second colour with food if the second

colour is presented within about 5 minutes of the initial training, while the

momory trace is in the short-term phase (Menzel, 1979). The second phase of

memory is called long-term memory. It is more persistent and lasts for days to

years (Carew et al., 1972; Pinsker et al., 1973). Once formed it is resistant to

interference from elentriconvulsive shock and conflicting experience. Short­

term memory requires covalent modifications of pre-existing proteins (no

protein synthesis is required) while long-term memory requires new protein

synthesis in all vertibrates and invertibrates tested so far (Goelet et al., 1986).

Short-term memory is suggested to be a part of an adaptive information­

processing strategy rather than a physiological necessary detour that the

information must take on its way to long-term storage. In a way short-term

m~mory is a way to temporarily hold information while waiting to see if it will

be corroborated (Erber, 1975a, b; Menzel, 1979, 1984).

12

The role of ocelli in insect behaviour has never been satisfactorily

established (Goodman, 1970). To explore the physiological function of ocelli,

we need to know the anatomy ofthe same (Fig. 1.3b). The external part of each

ocellus is a thick bi-convex lens. Beneath this lens is a layer of very simple

elongated retinal cells connected to nerve fibers which taper into the 'ocellar

nerve. An outer layer of long, parallel cells, perpendicular to the lens but

separated from the latter by a vitreous layer, constitutes the retina; which is the

light sensitive part of the ocellus. The retinal cells are arranged in groups of two.

or three and sometimes more on the ends of nerve branches, and each group is

termed a retinula. The opposing surfaces of the cells in each retinula secrete an

axial rodlike structure, known as a rhabdom, which probably serves to deflect

the light coming through the lens into the surrounding cells. The inner ends of

the retinular cells are produced into nerve fibers that traverse a mass of

interstitial cells behind the retina and go to the brain through the ocellar nerve

(Snodgrass, 1956). Fig. 1.3c depicts a transverse section of the median ocellus'

of an adult bee (Redikorzew, 1900).

It has been suggested that the ocelli might serve to increase the visual

field of the insect by either having a wider field of view than the compound

eyes or by viewing a part of the visual field unseen by the compound eye

13

(Goodman, 1970). Schricker (1965) found that the ocelli play an important role

in determining the onset and cessation of foraging activity. According to him,

the ocelli interfere in the foraging activity of the bees in the following way: the

light intensity required for the first and last collecting flight is increased by a

factor of 2 if one ocellus is covered, 3.3 if two ocelli are covered and 4.5 if all

the ocelli are covered.

Another behaviour, equally important as foraging, is defensive

behaviour. This open nesting bee is the most ferocious insect on earth (Morse

and Laigo, 1969). Honeybee colonies are well known for their defensive

posture when they are attacked by a predator or otherwise disturbed. Such

disturbance results in an organised colonial defense demonstrated by the colony

that follows a specific pattern of detection, alarm, recruitment, attack and then

stinging (Matschwitz 1963, 1964a, 1964b; Frisch 1967; Morse et al. 1967;

Collins et al. 1980).

The defensive response is elicited by alerted or stinging workers w:hich

release volatile alarin pheromones from their Koshevnikov's glands and the

setose membrane near the sting apparatus (isopentyl acetate) along with

mandibular gland secretions (2-heptanone) (Boch and Shearer 1962, 1966;

Boch and Rothenbuhler 1974; Koeniger et a!. 1979; Southwick and Moritz

14

1985). The intensity of defensive behaviour is dependent on external

environmental factors (aggressiveness is more under high temperature and high

humidity condition; under cool, overcast and windy conditions, aggressiveness

is less) as well as the genetic makeup of the colony (Schua 1952; Crewe 1976;

Collins 1981; Southwick and Moritz 1987b ). Attacking behaviour could be .

affected by the time of day also (Morse and Laigo, 1969).

On comparing the defensive behaviour of three Asian honeybees (A.

dorsata, A. jlorea and A. cerana), Seeley (1985) pointed out that A. dorsata is

highly defensive. This results from the two obvious reasons- it's large size

(weighing one and a half or two times as much as A. mellifera and three- five

times as much as A. jlorea or A. cerana), and its easily visible nest which

makes it more prone to attack by predators (Baroni Ubani, 1979). A dorsata

often constructs aggregations of nests in particularly favourable nest sites,

usually on large diameter smooth trees, rock overhangs or man - made

structures (Seeley et al., 1982). The fact that the A. dorsata nest aggregates are

within a few meters of each other provides an additional mechanism of

cooperative defense against large or persistent predators. There is always a very

little response from one isolated bee or a few bees and defensive behaviour is

dependent on the number of bees in the group (Southwick and Morse, 1985).

15

Another Asian honeybee A. cerana has developed specialized behaviour

to defend its nest from large Vespine predators. Instead of counterattack, they

form groups of about 30 individuals near the entrance, with the tips of their

abdomen raised (Schneider and Kloft, 1971 ). A sharp hissing sound is

repeatedly emitted from the hive after which the hornet usually leaves. Should

the hornet persist in the attack by attempting to capture a single bee out of the

group, all the A. cerana worker bees grab it's extremities and smother the hornet

in their own mass (Matsura and Sakagami, 1973) by shivering and thus

producing enough metabolic heat to kill the predator (temperatures in excess of

43°C kill a hornet but it can be tolerated by the bees).

Production of hissing sound (described as "Shimmering behaviour" by .

Butler in 1954) is evoked by various mechanical shocks such as a sudden blow

upon the hive, the abrupt opening of the hive lid etc., but occasionally without

any apparent external causes. When an A. dorsata nest is smoked gently, there

is first a rippling movement across the nest and then a low roar, unlike any

noise made by bees in the nest of A. mellifera (Morse and Laigo, 1969).

Lindauer (1956) has observed this first stage in alarm by A. dorsata. This bee is

more easily aroused under the unfavourable weather conditions or the vernal

and autumnal dearths, that is, situations in which A. mellifera colonies increase

16

their aggressiveness (Sakagami 1960). Sakagami (1960) found that the

repetition of the same shock raises the threshold to evoke this response. With

appropriate stimuli, shimmering is usually repeated 4-5 times and the time

interval between each shimmering lasts 3-5 seconds, often with a more

prolonged delay.

Bees shrug their wings along with the production of sound. The roar,

presumably made by bees moving their wings, is the second stage in the alarm

system (Morse and Laigo, 1969). With the wing stroke, bees push the body

forward, simultaneously without any locomotion. This communal reaction

appears as wave across the comb. Sakagami (1960) reported a momentary

quietness of the comb surface during this reaction as other regular activities like

walking, running or dancing ceases. Roepke (1930) and Butler (1954) described

similar behaviour in A. dorsata when disturbed by intruders such as hornets or

men.

Another type of behaviour related to non-attacking aggressive defense is

abdominal shaking (Sakagami, 1960). It refers to the repeated lateral

movement of the abdomen. The situations in which this reaction is observed are

i) in response to approaching strangers or nest mates (guards show this reaction

at the hive entrance), ii) if a hand or pencil etc. is shaken above the top bar of

17

~ -·- -

Fig. 1.5a. The Nasonov or scent gland (SntGl) in the abdominal region of an adult worker bee. Internal view of right half of abdomen of worker (A), underside of back

plate of abdominal segment VII showing scent gland (B). T indicates abdominal tergum.

Fig. I .5b. Position of the Nasonov gland (dorsal view) (Snodgrass, 1956), a is a line of strong submarginal inner ridge on the base of tergum VII of worker bee, b and c are

margins of elevation on tergum VII.

the comb (then bees of that area or vertically resting bees show this), iii) before

orientation flight, iv) occasionally during grooming dance.

If A. dorsata bees are provoked, unlike other Apis sp., they may attack

in large numbers. Attacking bees fly in a cloud usually 3 to 6 m in diameter,

most of them into open, sunny areas but some close to the ground, even in

shady places searching for intruders. The attacking force usually comes from

the mouth zone. The maximum attacking force probably includes not more than

10 percent of a colony population (Morse and Laigo, 1969).

All the worker bees of Apis possess the abdominal scent gland (Fig.1.5).

There is a pale area in between the 6th and 7th tergits (Snodgrass, 1956) which

actually, is (Jacobs, 1924) a weakly sclerotized basal part oftergum 7 to which

the intersegmental membrane is attached. Underneath this lies a mass of 500 to

600 scent gland cells. Noirot and Quennedey (1974) classified them as type III

glandular cells. The secretion of this gland contains seven volatile components:

geraniol, neralic acid, geranic acid, (E)- citral, (Z)-citral, (E-E)- famesol and

nerol. (Boch and Shaerer, 1962, 64; Weaver et al., 1964; Butler and Calam,

1969; Pickett et al., 1980; Winston 1991; Williams et al., 1981). Apart from

these volatile components, the secretion contains some soluble proteins (Zupko

et al., 1993). SDS- polyacrylamide gel electrophoresis indicats the presence of a

18

75 kDa doublet, a faint band at 60 kDa, a broad band at about 50 kDa, a faint

band at about 35kDa and some low molecular weight proteins. These volatile

components serve as pheromones and provide signals for swarmings, marking

of hive entrance, food source etc. Ferguson et a!. (1979) and Free et a!. (1984)

confirmed that the presence of the synthetic Nasonov pheromone was necessary

for swarm cluster formation. Traps with synthetic Nasonov lures increased bee

attraction by a mean of 114% over comparable traps without lures.

The following chapters deal with the different vision related

biological aspects of A. dorsata. The various aspects that are explored are the

visual memory of A. dorsata, the role of ocelli in colour or intensity

detection and defensive behaviour evoked by visual stimuli. In chapter lll

(part A) we· discussed how to train to come to a disered location using the

colour sense of bees. It seems that bees can retain the location of food source in

their memory. So we tried to find out whether there is any change in the

protein profile in the brain of the fully trained bees in comparisn with bees at

the early stage of training (chapter lll- part B). Colour receptors had been

reported in the ocelli of honey bee. We checked whether ocelli of Apis dorsata

has any direct role in colour ditection (chapter lV- part A). Role of ocelli in the

intensity detection was carried out in chapter IV- part B as light intensity and

19

foraging behaviour is directly related. Aggressive defensive behaviour of this

bee evoked by visual stimuli has been discussed in detail (chapter ,V). It has

been observed that the defensive behaviour of A. dorsata is associated with

the scent gland (Nasonov gland). The structure of this gland and the

chemical composition of its secretion have also been studied (chapter Vl).

20