Melissopalynological Analysis of Honeys from Paderu Forest Division of Visakhapatnam District in Andhra Pradesh, India

Palynological examination of 17 honey samples procured from 8 localities in Paderu forest division in Visakhapatnam district, Andhra Pradesh, India, produced assemblage of pollen in terms of quantity and diversity. According to melissopalynological assessment of the honey samples, 6 were unifloral, i.e., 3 from Ageratum conyzoides and 1 each from Schleichera oleosa , Psidium guajava , and Mimosa pudica , and 11 were multifloral. The dominant taxa include Mimosa pudica , Syzygium cumini , and Centipeda minima . The taxa such as Terminalia arjuna , Dendrophthoe falcata , Lamiaceae , Asteraceae , and Phyllanthus emblica were minor sources of nectar and bee forage, as indicated by low frequencies of their pollen. The numerous pollen types and their diversity show that bees travel considerable distance to collect the nectar for honey production.


Introduction
Melissopalynology is a branch of plant sciences that studies pollen found in honey. Precision in interpreting pollen data recovered from the honey has always been a primary goal of those who study pollen and honey. We are using pollen count to determine the nectar source of a honey sample and recognize the types and percentage of recovered pollen in the honey. This study is the fact that honey bees utilize certain natural raw materials which are identifiable in honey. These natural raw materials include pollen and nectar [1]. The growth and development of honey bees depend on nectar as the source of carbohydrates and pollen as the source of proteins [2,3]. Palynological analyses of honey and pollen loads are used to know about honey bee foraging ecology, habitat and vegetation [4][5][6][7][8][9][10][11]. Pollen of honey samples provides reliable information on floral resources of honeys along with the relative preferences of bees among the diverse assemblages of plant species flowering synchronously [12,13].
The significant melissopalynological works have been reported from different sectors of this state, dealing with pollen analysis of honey [14][15][16]. Similar melissopalynological research work has been worked out in Karnataka [17][18][19],

Materials and methods
The materials for the present study are 17 honey samples (50 ml each) that were procured from 8 different blocks of Paderu forest division during 2011-2013, i.e., 3 samples from Ananthagiri (Af-13, Af-34, and Af-57), 4 from Paderu (Af-10, Af-40, Af-44, and Af-64), 2 from Munchingiputtu (Af-14 and Af-54), 2 from G.Madugula (Af-3 and Af-48), 3 from Peddabayalu (Af-11, Af-53, and Af-65), and one each from Araku Valley (Af-37), Dumbriguda (Af-9), and Hukumpeta (Af-51). For palynological assessment, the honey samples were chemically processed using the acetolysis method, i.e., 1 ml of honey sample was dissolved in 10 ml of distilled water and centrifuged. The supernatant liquid was drawn out. The resultant sediment was treated with 5 ml of glacial acetic acid and centrifuged. After decanting acetic acid, the sediment was treated with acetolysis mixture (5 ml). It was prepared by nine parts of acetic anhydride and one part of con. sulfuric acid and then heated under water bath until the liquid turned chestnut-brown color. After cooling it was again centrifuged and the supernatant liquid was decanted. The sediment then treated with glacial acetic acid, later centrifuged, and the supernatant liquid was decanted off. And the sediment was washed with distilled water, and 50% aqueous glycerin (5 ml) was added and centrifuged for 10 min. The supernatant liquid was decanted off, and the tubes were inverted upside down on a filter paper for a few minutes [34].
The pollen sediment was taken on a pellet of glycerin jelly and transferred to the center of the slide. After being warmed slightly, the melted jelly with pollen sediment was covered by cover slip. Cover glass was later sealed with paraffin wax and labeled with their respective codes. Three slides were prepared for each sample and studied critically for their pollen contents.
Identification of pollen recovered from honey samples were carried out through the consultation of reference pollen slides available in Paleobotany and Palynology lab, UCS, Saifabad, OU. Quantitative pollen analysis was based on the method recommended by the International Commission for Bee Botany [6]. Pollen contents were taken at random, covering the maximum mounted area to avoid repletion. Once identified and counted, the pollen grains were placed into one of following pollen frequency classes-predominant pollen types (>45%), secondary pollen types (16-45%), important minor pollen types (3-15%), and minor pollen types (<3%). Honey samples containing more than 45% of a single type of pollen were considered as unifloral honey. The pollen types were placed into arboreal and non-arboreal taxa for making honey pollen spectra (Figure 2). A detailed list which included sample number, locality, nature and type of honey, collection season, and frequency of pollen types recovered is given in Table 1.
Hukumpeta (V-P-HP-R-AF-51, spring collection): the sample is palynologically proved to be multifloral as evidenced by secondary pollen types, Eucalyptus globulus

Discussion
The present Melissopalynological study provides new insights into the pollen composition of honey samples from Paderu forest division in Visakhapatnam district of Andhra Pradesh. A total of 69 pollen morphotypes from 17 honeys produced by Apis florea were identified. Six honeys were considered unifloral honeys because they contained a predominant pollen type (>45%). The dominant of unifloral honeys, without any toxic pollen grains and with scarce fungal elements, suggests that most of the honeys are of good quality and suitable for human consumption.
The results coincide with the melissopalynological investigation in the peninsular part of India where unifloral honeys are dominant [35]. The diverse flora of India is due to varied climatic conditions in different parts of India. The multifloral source of honeys may be generated by the absence of major ingredients of forest and invasion of secondary forest elements [36]. The Palynological analysis of Paderu forest division honeys reflects that the native flora may be used as a source of good quality honey. In our studied honey-pollen exploration, it is easy to perceive that the honey bee preferred mainly non-arboreal in spring honeys, with the exception of Af-37 as arboreal dominant; Ageratum conyzoides is a predominant pollen taxon in three spring samples, Mimosa pudica, Tridax procumbens, and Conyza stricta as secondary pollen taxa. In autumn season also, bees preferred mainly non-arboreal exception of Af-13 and -64 samples, in Af-13 sample as unifloral with Schleichera oleosa as predominant pollen taxa.
Based on the above study, bees are preferred mainly on non-arboreal to collect nectar and convert to honey due to the flowering time of the melliferous species; climatic condition and human activities (e.g., farming, reforestation, and forest fires) may be other factors to consider in understanding the presence or absence of some taxa in the pollen spectra of honeys. In recent years, many rural communities have taken up beekeeping as an alternative source of their livelihood strategies. Even the younger generation is showing interest because beekeeping is so easy and simple that anybody can take it as an enterprise. Thus, our melissopalynological investigation may contribute to and favor the possibilities of using rich flora of the studied area in order to develop beekeeping enterprises on a commercial basis, in which self-employment opportunities may be created for many rural communities and develop their livelihood strategies in this area.

Conclusions
The analysis of the pollen content of Paderu forest division in Visakhapatnam district of Andhra Pradesh honey samples indicates that the local flora may be used as a source of good quality honey. The overall preponderance of non-arboreal in most of the honeys reflects that the honey bees prefer to visits to collect nectar. The scarce appearance of pollen from nectar-less plants such as Amaranthaceae, Cyperaceae, and Poaceae indicates that they were trapped in the hive incidentally by wind or were inadvertently transported by honey bees. And apiculture may enhance honey production in floristically rich province of Paderu forest division in Visakhapatnam district of Andhra Pradesh and adjoining areas, when job opportunities may be created for many developing rural communities of this state.