Characterization of microbiota associated with stingless bee Tetragonula travancorica Shanas and Faseeh(Hymenoptera:Apidae:Meliponinae) and its pollen

Abstract

Stingless bees are small to medium-sized eusocial insects belonging to the tribe Meliponini (Hymenoptera: Apidae). They are one of the potential pollinators in the tropics and adapted to varied climatic conditions than other pollinators such as honey bees and bumble bees. There are over 600 species of stingless bees worldwide, with India recording three genera viz., Tetragonula Moure, Lisotrigona Moure, and Lepidotrigona Schwarz. Tetragonula is the most complex and widely distributed genus with more than 30 and 17 species worldwide and in India, respectively. A new species was discovered in Kerala, Tetragonula travancorica Shanas and Faseeh, and found to be the widespread species of stingless bee in peninsular India. Stingless bees and their products host symbiotic microbes, which play a prominent role in metabolism, hormonal signaling, behaviour, gut physio-chemical conditions, growth and development, protection against pathogens, and the immune response of bees. However, the microbial communities associated with stingless bees and their pollen remain poorly understood. Hence, the present research programme was proposed to study the pollen sources, and microbiota associated with stingless bees, hive-stored pollen, and flower pollen. A survey was carried out to collect stingless bees (T. travancorica) and their hive-stored pollen from all districts of Kerala for palynological and microbial diversity analysis. Palynological studies conducted through light microscopy and scanning electron microscopy revealed the presence of 102 pollen types from 38 plant families. Eight predominant pollen types were found across Kerala, viz., Mimosa pudica, Mangifera indica, Coffea arabica, Bauhinia purpurea, Alternanthera sessilis, Pennisetum polystachion, Artocarpus heterophyllus, and Cocos nucifera. The hivestored pollen analysis of stingless bee T. travancorica recorded the highest number of pollen types from the family Fabaceae (14) followed by Asteraceae (9) and Euphorbiaceae (6). The maximum pollen types belonged to the trees followed by weeds, ornamentals, and horticultural crops. Absolute pollen count was the highest in Kannur district (11,01,000 pollen grains/mL) and lowest in Idukki district (1,28,500 pollen grains/mL). The microbial diversity analysis of hive-stored pollen revealed the presence of bacteria and fungi, whereas, yeast and actinomycetes were not detected across Kerala. A total of 24 bacterial and 20 fungal isolates were identified in the hive-stored pollen and their morphological, and cultural characteristics were recorded. Among them, distinct bacterial isolates were subjected to biochemical characterization, which revealed that all bacterial isolates showed positive for the catalase test, while six isolates were positive for the oxidase test. All the isolates except one isolate were negative for the methyl red test and five isolates were positive for the Voges Proskauer test. Seven isolates showed positive reactions for sucrose and fructose fermentation, which helps in identifying bacterial isolates. Molecular characterization of bacterial isolates revealed that Bacillus spp., B. cereus, B. acanthi, B. velezensis, B. subtilis, B. megaterium, Priesta aryabhattai, Pseudomonas spp., P. aeruginosa, Klebsiella pneumonia, and Acinetobacter spp., were recorded in the hive-stored pollen of stingless bee. Fungal isolates viz., Aspergillus flavus, A. aculeatus, Penicillium spp., and P. chrysogenum were identified in the hive-stored pollen of stingless bees. The microbial load to pollen grain ratio was highest in Idukki (1:30.5) and lowest in Kollam (1:25750). The highest total bacterial and fungal population was observed in Idukki (4160 cfu g-1 of pollen), Pathanamthitta, and Thrissur (90 cfu g-1 of pollen). The lowest bacterial and fungal population was recorded in Kollam and Alappuzha (10 cfu g-1 of pollen), respectively. The predominant pollen and secondary pollen obtained from the various districts of Kerala were selected to study flower microbial diversity. The microbial analysis of flower pollen revealed the presence of bacteria and fungi, whereas yeast and actinomycetes were not detected in the flower pollen. A total of 19 bacterial and 21 fungal isolates were recorded in the flower pollen and identified with morphological and cultural characteristics. Biochemical tests revealed that all the isolates were catalase-positive and four being oxidase-positive. Six bacterial isolates tested positive for the methyl red test, while five were positive for the Voges- Proskauer test. Eight isolates showed sucrose utilization, indicated by a color change from red to yellow without air bubbles in the durum tube. Molecular characterization of the bacterial isolates revealed that Priesta aryabhattai, Bacillus megaterium, B. safensis, B. pumilis, Pseudomonas aeruginosa, Pantoea spp., P. dispersa, and Serratia marcescens were present in the flower pollen. Similarly, molecular characterization of fungal isolates revealed that Penicillium spp., P. chrysogenum, P. citrinum, Curvularia warraberensis, C. clavata, Aspergillus spp., A. niger, A. aculeatus, A. flavus, Pestalotiopsis spp., Fusarium incarnatum, and Mucor irregularis were present in the flower pollen. The microbial load-to-pollen grain ratio was highest for Pennisetum polystachion (1: 0.84), and the lowest ratio for Mimosa pudica flower pollen(1:8.8), Alternanthera sessilis had the highest total bacterial population (63,000 cfu g-1 of pollen), while Mimosa pudica had the lowest (5000 cfu g-1 of pollen). Similarly, Areca catechu had the highest total fungal population (90 cfu g-1 of pollen), while Pennisetum polystachion had the lowest (30 cfu g-1 of pollen). The gut microbiota of stingless bees, T. travancorica, and their hive-stored pollen were investigated and compared through metagenomic analysis under a nextgeneration sequencing platform. The sequence analysis revealed that Lactobacillus (41.57 %) was the dominant symbiont in the gut of T. travancorica, while Bacillus (28.11 %) was the predominant genus in the pollen of T. travancorica. Similarly, the fungal symbionts associated with the gut of T. travancorica revealed that Rhodosporidiobolus (15.46 %) was the dominant fungal symbiont in the gut, while Saccharomycopsis (27.67 %) was dominant in the pollen. Characterization of the microbiota associated with the stingless bees and their pollen unveils the complex relationship between bees and their microbial symbionts. Bee and its products are potential sources of beneficial microorganisms that have wide applications in various fields. The specific microbiota associated with the bees helps to enhance beekeeping practices, reducing colony losses and aiding in the conservation of these crucial pollinators. Harnessing these bees and their symbionts has an immense scope in agriculture, food, and probiotic industries