Microbial diversity in hive-stored pollen of indian honey bee, Apis cerana indica (Fabricius)

Abstract

Honey bees are eusocial flying insects. Among all insects that pollinate, honeybees are considered to be one of the crucial pollinators. Honey bees represent only a small fraction of the roughly 20,000 known species of bees. Honey bees obtain all of their nutritional requirements from a diverse combination of pollen and nectar. Pollen grains referred to as 'bee bread' or 'bee meat' are the natural sources of proteins, vitamins, fats, lipids and minerals, of which proteins are very much essential for building the body tissues of the bees, especially during the early embryonic growth. Worker bees of Apis cerana indica help in the fermentation of pollen which releases additional nutrients that are used in the production of antibiotics and fatty acids which inhibit spoilage. Pollen analysis of samples provides the information regarding the plants preferred by bees for nectar, as the pollen grains dispersed are mostly collected by bees along with nectar. Honey has two sources of contamination, primary one being the hive stored pollen. Thus, it is very essential to study the microbial diversity of pollen, which may lead to spoilage of pollen and nectar. The present investigation on “Microbial diversity in hive-stored pollen of Indian honey bee, Apis cerana indica (Fabricius)” was conducted at Department of Agricultural Entomology, College of Agriculture, Vellanikkara, Kerala Agricultural University during 2020-2021 in order to study the diversity of hive-stored pollen and associated microbes in A. cerana indica (Fabricius) colonies. Purposive surveys were conducted in six locations at Palakkad and Wayanad district viz., Mannarkkad, Nellipuzha, Nottaamala, Kenichira, Kayakunnu and Cheengode. Hive stored pollen collected were maintained in refrigerated conditions after assigning unique accession numbers as PKD-1, PKD-2, PKD-3, WYD-1, WYD2, WYD-3. Microbial diversity, microbial load of hive stored pollen, identification and characterization of pollen with respect to its nature, size, shape, aperture, exine pattern along with physicochemical properties viz., moisture content, water activity, pH, ash content, total acidity and protein content were studied. Palynological identification plays a crucial role in beekeeping industry. Pollen aids as a food supplement to honeybees for their growth and development. It assists in the identification of geographical and botanical origin of pollen. A total of 21 plants were recorded as pollen sources by bees from the study area (six locations) of two districts. Ten pollen types were identified based on its nature, size, shape, aperture, exine pattern using PalDAT software. Identified plant species were Sphagneticola trilobata (Asteraceae), Caesalpinia pulcherrima (Fabaceae), Zinnia elegans (Asteraceae), Ocimum sanctum (Lamiaceae), Clitoria ternatea (Fabaceae), Biophytum sensitivum (Oxalidaceae), Bauhinia acuminata (Fabaceae), Mimosa pudica (Fabaceae), Tecoma stans (Bignoniaceae), Portulaca grandiflora (Portulacaceae). Characterization of pollen revealed that ten plant species belonging to seven families serve as pollen sources in respective survey locations. Physicochemical properties of hive stored pollen of A. cerana indica collected from six locations were investigated by estimating the moisture content, water activity, pH values, ash content, total acidity and protein content. Moisture content varied from 15.29-15.38 (g/100g). Water activity varied from 0.73 to 0.74 (aw) and the pH varied between 3.77 and 3.83. Ash content varied from 1.87-2.01 (g/100g). Total acidity varied from 9.64-10.13 (mmol/g). Protein content varied from 3.46-3.69 (%). Parameters like moisture content, water activity, protein content were more in the Wayanad sample, it may be because of high humidity and temperature variations. Parameters like pH, total acidity and ash content were more in Palakkad sample, which infers that mineral content might be more. Microbial diversity of hive stored pollen of Apis cerana indica collected from six locations was investigated by identifying the microbes through their morphological, cultural, molecular characterization. Other parameters like microbial diversity, microbial load, microbial load to pollen grain ratio, microbial population were also observed. In total, four bacteria and three fungal species were identified. However, colonies of actinomycetes and yeast were not obtained. Out of four bacteria, B2, B3, B4 – gram positive while B1- gram negative. The shape of all bacterial isolates was rod with change in their colony colour.16s rRNA sequencing of bacterial isolates revealed that B1 as Pseudomonas aeruginosa, B2 as Bacillus megaterium, B3 as Bacillus aryabhattai and B4 as Bacillus megaterium respectively. Out of the three fungi, colony colour of T1 colony colour was green, W1 colony colour was white and A1 colony colour was orange. All isolates have flat elevation with an entire margin. ITS sequencing of fungal isolates revealed that T1 as Trichoderma reesei, W1 as Westerdykella multispora and A1 as Neurospora crassa. Microbial diversity was more in sample collected Wayanad with two fungal and two bacterial colonies whereas, one fungal and two bacterial colonies in sample collected from Palakkad. Microbial load was more in samples from Wayanad (6.6x104 cfu/g) when compared with Palakkad sample (3.3x104 cfu/g). Comparison of microbial load to pollen grain ratio revealed that Wayanad sample recorded highest with 6.6x104 cfu/g to Palakkad sample with 3.3x104 cfu/g. Bacterial and fungal populations were observed more in Wayanad sample when compared to Palakkad sample. The study recorded the pollen sources of different localities in and around the Palakkad and Wayanad districts of Kerala. Moisture content plays a significant role which might be the reason for high microbial diversity and microbial load in Wayanad. The present study demands the investigations of other physicochemical properties like EC, starch content, amino acid composition, microbial spores present in pollen which could be useful for the food preservation industry or potentially hazardous in due course of time.