The below mentioned article provides a study note on the melissopalynology.
Melissopalynology or melittopalynology [respectively also spelt as mellissopalynology or melittopalynology. International Commission of Bee Botany (ICCB), Louveaux et al, (1978) spelt as mellissopalynology. So in this essay the spelling of ICCB is followed] is the study of pollen and spores present in honey.
By extension, it also includes honeydew elements (HDE), i.e. fungal spores, hyphae and microscopical algae and other bioparticles. ‘Melissa’ and ‘melitta’ mean a bee according to Paxton’s Botanical Dictionary, 1868. Apis mellifera is the scientific name of common honeybee. In Latin the word mellifera means honey. In addition to Apis mellifera L. the other Indian honeybees are A. indica F., A.florea F. and A. dorsata F.
Honeybee requires nectar, pollen, resin and water for their survival. The bees are adopted for feeding on nectar and pollen. Resin reinforces the hive and water cools the hive. Moreover water dilutes the honey that is fed to larvae. In a hive there are three types of bee: the single queen bee, a variable number of male bee or drone and 20000 to 40000 or more worker bees.
The worker bees travel long distances to collect nectar. They suck nectar from nectary present in the flower. The nectar is stored in the crop of bee. Regurgitation of nectar starts before the return of bees. On return to the hive the other worker bees remove the nectar. The foraging bees and other worker bees regurgitate the nectar.
Regurgitation occurs a number of times till the nectar is transformed into honey. During the process the natural complex sugar (sucrose) of the nectar is converted to simple sugar (fructose, glucose etc.) by the enzymes. The sugary fluid is then stored in honeycomb, which is left unsealed. The honeycomb is a group of hexagonal cells composed of beeswax and propolis.
The sugary fluid is high in water content. The formation of ripe honey is accomplished by evaporating the excess water. This happens when the bees inside the hive fan their wings thus circulating air across the honeycomb. The reduction in water content causes the increase of sugar concentration. The honeycomb containing the ripe honey is then capped (sealed) with beeswax and propolis.
Honey is thick, sweet syrupy, white or yellowish or brown or other colours, and viscid fluid. Honeybees from the nectar of flowers produce it. Honey is consumed primarily as an energy source and is stored in the honeycomb for unfavourable situation like winter etc. Pollen grains are used as the primary source of protein and other nutrients.
Honey is composed of 14-18% of water, 76-80% of glucose, fructose, levulose, dextrose etc., pollen, mineral salts (compounds of calcium, potassium, sodium, magnesium, copper, silica, phosphorus, manganese, silicon, iron etc.), acids (malic, tartaric, citric, succinic etc.), plant pigments like carotene, xanthophyll and chlorophyll; enzymes like invertase, diastase, catalase etc., vitamins (B1, B2, K, folic acid, biotin, pyridoxine, pantothenic acid etc.) and amino acids (cystine, lysine, glycine, aspartic acid, glutamic acid, alanine, valine etc.).
The composition varies and it is dependent upon the flower that provided the nectar. The variation in colour is also dependent on the plant that foraged the bee, i.e. alfalfa and clover produce white honey and Acacia produces straw colour honey. The honeys produced from cotton, Plectranthus and Eucalyptus are also white.
Golden or light yellow coloured honeys are obtained from litchi, mustard and rubber. Honey is significantly sweeter than table sugar. It was almost the sole sweetening agent from ancient times until cane sugar became commercially important. Honey being complete natural and biological product and free from any type of impurities, it is often preferred over sugar.
It is one of the best health food consumed by man. Honey has become a common ingredient in many types of ayurvedic medicines as it is found to contain certain types of medical benefits. Apart from food, honey has always been held in high regard. In language, literature and culture honey is frequently used as a symbol that is supposed to bring good luck or to keep away evil. It is the symbol of sweetness of every kind.
The honey does not spoil easily. Due to the presence of low water and high sugar concentration, it does not allow to grow any airborne yeast or bacteria. Yeast/bacteria get plasmolyzed when they come in contact with concentrated sugary honey. So long the moisture content remains below 18% almost no microorganisms can multiply in honey.
Pollen grains are collected during the visit of plants by bees for nectar. The legs of bee help in this process. The legs are not simple structure to walk on. They are highly specialized organs that enable the bee to collect pollen. The legs are present on the thorax of a bee. The bee thorax consists of three body segments and six legs, one pair of legs being present per thoracic segment (Fig. 5.1 A).
Each pair of legs has the same construction, i.e. consists of coxa, trochanter, femur, tibia and tarsus. The tarsus consists of five sub-segments —one elongated basitarsus and four smaller tarsomeres.
The basitarsus has pollen brush on the inner side. The hind legs of worker bees are highly modified to carry pollen and propolis. The tibia of worker bees has corbicula, commonly called pollen basket, on the outer surface of tibia (Fig. 5.1B).
The pollen basket is an expanded, slightly concave region of tibia. Its surface is smooth. A stiff row of bristles borders the inner edge of tibia, called pollen rake or rastellum. The basitarsus of worker bees has pollen combs on the inner side that consist of a regular series of stiff bristles (Fig. 5.1C).
The base of basitarsus has a flattened area called pollen press. The above mentioned structures are meant for manipulating pollen into the pollen basket. During foraging pollen grains stick to head, abdomen and other body parts. The bee moistens the forelegs with the protruding tongue and collects pollen grains from the front body.
Pollen grains from the forelegs are then transferred to middle legs with the help of pollen brushes. The pollen grains now present on the middle legs are passed to the pollen combs present on the inner basitarsus of inner hind legs. This happens when pollen combs scrape pollen from the middle legs and abdomen.
Then pollen grains pass to pollen basket of opposite hind legs. This occurs in a very ingenious way. The pollen rake scrapes the inner surface of each opposing pollen comb (Fig. 5.2). As a result pollen grains come to lie on pollen press of opposite leg. Pollen grains thus accumulated over the press are pushed into the pollen basket by straightening the hind leg.
As a result pollen grains are pressed, compacted and squeezed into the pollen basket’. Pollen-collecting-bees can be recognized instantly by conspicuous balls of bright-coloured pollen packed onto their hind legs. All the above manipulations regarding the collection of pollen and deposition of pollen in pollen basket are carried out at brief intervals.
Thus the pollen basket is gradually filled with pollen. The basket is also filled with pollen mixed with nectar and regurgitated nectar. The compressed pollen content in the pollen basket is known as pollen pellets. A microscopical study of pollen grains present in pollen pellets will reveal the plant species that the bee visited during foraging.
Honeybees also collect honeydew instead of nectar. Honeydew is a sugary substance secreted by aphids and other plant-sap-sucking insects. Honey is also produced from honeydew by bees and is known as honeydew honey.
It consists water (16%), fructose (38%), glucose (27%), sucrose (7%), dextrose (9%), acids (7%) and minerals etc. The following plants are the hosts of aphids and other insects that produce honeydew-Abies alba, Acer, Corylus, Fagus, Fraxinus, Carya, Larix, Malus, Pinus, Prunus, Quercus, Salix alba, Sorbus aucuparia, Ulmus americana etc.
Melissopalynology is an early branch of palynology (the study of pollen and spore). Pollen analysis of honey or melissopalynology has great importance because it reveals the plant species foraged by honeybees. The identified plants provide the fingerprint of the environment and pollen spectrum from where the honey comes.
Pollen analysis also provides the botanical and geographical origin of honey, information about honey extraction, presence of contaminants like rust and smut spores, kinds of adulterant etc. Today pollen analysis has become an indispensable tool to authenticate honey.
Microscopical analysis is the prerequisite to determine the pollen spectrum of a honey sample. For microscopical examination the honey sample is diluted with water and the microscopical elements are concentrated by centrifuging.
The elements are studied under microscope either after acetolysis (it is a technique used in the preparation of slides of pollen grains and spores to study the morphological details specially the sporoderm stratification) or without further treatment.
The procedure of acetolysis is usually omitted to determine HDE, thin- wall-pollen grains and other bioparticles as they get dissolved during the process. For the identification of pollen grains acetolysis is a must because palynological literatures provide the identifying characters from acetolysed materials.
Slides are prepared for microscopical examination. Louveaux et al. (1978) and von Der Ohe et al. (2004) published the details regarding the method of preparation of slide and counting methods of pollen. In a nutshell, the sediments after centrifugation (acetolysed/without any treatment) of a honey sample are spread evenly with a micro spatula over an area of 22mm × 22mm on the slide.
For this purpose a square of 22mm × 22mm was drawn on the back of microscopical slide with a permanent marker to prevent unintentional dyeing of pollen and discolouration of the markings. The spread was mounted in glycerine jelly and covered with cover slip of 22mm × 22mm or other appropriate size.
The microscopic analysis of the honey sample includes the identification of pollen grains and other microscopic constituents (e.g. HDE), quantification and determination of frequency classes and their percentages. A collection of reference slides is indispensable for the identification.
The reference slides may be acetolysed or unstained. In the latter case the pollen may be degreased or without the removal of fat. It is recommended to collect mature buds instead of open flower and letting them to open in the laboratory for preparation of slides to avoid contamination from anemophilous pollen.
In a honey sample pollen of different plant species may be present as the worker bees visit different plants. In addition anemophilous pollen may be incorporated into honey. Airborne pollen grains (ex. Gramineae, Cyperaceae, Rumex, Cannabis, Quercus, Amaranthus, Abies, Pinus, Picea, Larix, Alnus, Betula etc.) enter the hive on wind currents.
Moreover airborne pollen grains deposit when beekeeper removes honey from a hive. A honey sample may also contain honeydew elements like spores of Uredinaceae, Ustilaginaceae, Peronosporaceae etc.
Pollen from non-nectariferous and more or less entomophilous (ex. Papaver, Plant ago, Thalictrum, Chenopodiaceae, and Artemisia etc.) may also be present in a honey sample. Sometimes bees visit these plants for pollen collection.
After the identification of pollen grains in a honey sample, it is to estimate the relative frequencies of pollen type. It is necessary to count 1000 pollen grains for the determination of relative frequencies.
The sequence of counting is illustrated in Fig. 5.3. Pollen of wind-pollinated or nectarless plant, pollen of nectarless but more or less entomophilous plant, HDE consisting of fungal spores and hyphae, algae and plant-pathogenic fungal elements, e.g. spores of Uredinaceae and Ustilaginaceae, Peronospora etc. are to count separately.
The terms used in interpreting the counts and estimating the pollen grain frequencies and their frequency classes are illustrated in Table 5.1.
After analyzing the pollen and HDE contents it is possible to deduce the pollen spectrum of a honey sample. This can be verified with pollen spectrum of the declared botanical and geographical origin of honey. For the determination of botanical origin the frequencies of pollen type are calculated on the nectariferous species only. The pollen grains from the anemophilous and nectarless plant are excluded from the count.
The determination of botanical origin is possible for those honeys that are extracted centrifugally. Sometimes honey is filtered through diatomaceous earth or similar materials to remove pollen and other particles. In this case due to lack of pollen the botanical origin is to be verified by other method. The honeydew honey contains higher percentage of pollen from nectarless plant than that of flower-honey.
The honeydew honey also contains more HDE than pollen (P), the ratio being HDE/P > 3. The geographical origin of honey can be verified by comparing with pollen spectrum of that region.
In some instances the geographical origin can be determined by the presence of characteristic pollen of that region, e.g. the presence of Banksia pollen indicates Australian origin; the presence of Roystonea pollen is the geographical marker of Cuban honey etc.
The pollen spectrum of honey depends on the local vegetation where it was produced. Political boundaries are rarely associated with it. So microscopical analysis and interpretation indicate the geographical provenance of honey rather than the country of origin.
Honeys are of two types — unifloral honey and multifloral honey. In the former the bees collect nectar from one particular source plant, e.g. heather honey, citrus honey etc. In multifloral honey the bees collect nectar from different source plants e.g. honey sample obtained from greater Kolkata contains the pollen grains of Cassia, Cocus, Solanum, Tridax, Borassus, Croton, Polygonum, Chenopodiaceae etc.
The important honey plants of Jammu & Kashmir are Mesembryanthemum, Adhatoda vasica, Celosia, Impatiens, Tecoma, Opuntia, Cassia, Helianthus etc. In Kashmir region the following honey plants are present: Salix, Prunus, Pyrus, Juglans, Rosa macrophylla, Cucumis, Frageria, Iris, Robinia etc.
In unifloral honey it is frequently observed that the pollen grains are over- represented, i.e. the percentage of pollen in honey is greater than the percentage of corresponding nectar. Ex. Castanea (>80%), Brassica (>60%) etc. The reverse situation is also observed where the percentage of pollen in nectar is greater than the percentage of the corresponding honey; in this case the pollen grains are under- represented.
Ex. Citrus (10-20%), Medicago (20-30%) etc. In unifloral honey the percentage of single pollen type is 45% or more. A single pollen type with 45% concentration is the minimum requirement for a honey to be classified as unifloral. The over-represented type fulfills the above criterion only. There is no value to determine the under-represented pollen type.
Later the idea of absolute pollen concentration (APC) of a particular flower type was developed. In this concept a pollen coefficient class expressed as an average number for a particular flower type was deduced to determine the unifloral honey sample (under – represented or over – represented) regardless of data revealed by calculating the percentage of relative pollen concentration.
The average number of pollen coefficient class was obtained in the following way. A small number of caged honeybees were allowed to feed on flowers of a particular plant species. The honey thus produced contains the pollen grains of that particular plant species. It is expected that this honey sample is the valid representation of absolute pollen concentration (APC) for that particular flower type.
Different plant species of unifloral honeys were used as experimental material and in each case an average number of APC is deduced. These data were illustrated in a tabular form called pollen coefficient table. These data are used to determine the unifloral nature of honey in question.
Unifloral honey is always preferred over multifloral honey. The former is considered as the premium type and has high demand. Microscopical analysis determines the botanical and geographical origin of the premium honey.
But the verification of the source plant often becomes difficult because many samples have pollen grains that are traditionally under-represented. So pollen coefficient values are currently being used throughout the honey industry to verify the premium honeys.
From ancient times beekeeping was traditionally practiced for bees’ honey harvest. The other major hive products are beeswax, royal jelly, propolis and pollen.
Beeswax is the byproduct of the honey making process. The young worker bees secrete it. The bees have eight wax-producing glands on inner side of the abdomen. These glands secrete wax in the form of thin scales. Bees use the wax to build the honeycomb and to seal the opening of the filled honeycomb cells.
The honeycomb is the site of storage of pollen and honey, and of raising the young bees. The colour of beeswax varies from nearly white to brownish. It is usually yellowish and the colour is dependent upon the plant species that the bees visit. Beeswax is used for the production of candles, furniture polish, art materials, cosmetics, drugs etc.
Royal jelly (RJ) is the secretion of hypopharyngeal glands of bees. It is a creamy, milky white, acidic with high nitrogenous substances. It tastes bitter and has slight pungent smell.
It is to feed the queen bees throughout their larval and adult lives. Though it is not derived directly from plants RJ contains pollen grains coming from the foraging activity of honeybees. So pollen analysis of RJ will reveal its geographical origin and the environment where the beehive is located.
Royal jelly contains B-complex vitamin, water, several amino acids, simple sugars, fatty acids, trace minerals, enzymes, antibacterial and antibiotic components, and trace amount of vitamin C. Bees use RJ because it has the potential utility as food for rearing queen bees. People use RJ as dietary supplement as it has medical benefits.
Propolis is a sticky, gummy and resinous substance. The bees collect it from the buds and barks of trees. In the hive it is mixed with beeswax, enzyme and pollen.
Bees use porpolis:
(i) As cementing material to cement the honeycomb cracks thus reinforcing the structural stability of the hive,
(ii) To seal the alternate entrance thus making the hive more defensible,
(iii) As a disinfectant, and
(iv) To protect colonies against bacteria, putrefying corpuses, and unpleasant odour etc.
The colour of propolis varies from green to brown and reddish. It depends on its botanical source. The composition of propolis also varies between hives, seasons and places of origin.
In general it consists of resins and vegetable balsam, beeswax, essential oils, pollen grains, amino acids, vitamins, salts of calcium, potassium, zinc, iron etc. It is sticky at room temperature. It becomes hard and brittle at lower temperature. Propolis has medical benefits and is mainly used as disinfectant.
Pollen grains are the source of vitamins and proteins to the bees. It is highly nutritious and is used as dietary supplement. To collect it, the beekeepers force the bees to fly through meshes (also called pollen trap) where the openings scrape the pollen from the legs of bees. The pollen grains are dropped in a container from where they are collected.
There are certain plants where pollen are toxic to bees and the cause of bee poisoning, i.e. Rhododendron, Kalmia, Corynocarpus, Zygadenus, Cuscnta, Aesculus, Tilia, Solanum etc. In case of pollen poisoning dead bees are found near the plants that the bees visited while in pesticide poisoning dead bees are found near the hives.
Honey is not always edible. It may contain allergenic pollen and poisonous pollen grains, e.g. pollen of Datura, Rhododendron etc. Moreover the bacterium Clostridium botulinum can survive on honey and cause botulism in infants when fed.
Lastly it is to mention that the correctness of pollen identification, their quantification and determination of relative frequencies, and the subsequent interpretation of results of a honey sample form the basis of melissopalynological analysis.