Study On The Microbial Diversity Of Bee Pollen And Bee Bread By Apis Cerana And Apis Mellifera

Bee pollen,which is composed of plant pollen,nectar and secretions from bee glands,is a kind of cluster carried home by the "pollen basket",a special organ on the hind limbs of bees.Bees store bee pollen in their nests.Under the action of microbial fermentation,bee pollen is gradually transformed into bee bread that can be stored for a long time and is easy to digest.It is a source of protein for the larvae and all other stages of development of bees.In order to improve the productivity of bees,we should find the beneficial microorganisms from bee pollen and bee bread,and use the beneficial microorganisms to improve the nutritional health of bees.Bee pollen and bee bread contain a large number of probiotics,which is an important source of probiotics for the research and development of bee feed and fermentation of human food.However,there is currently a lack of systematic research on the diversity of bacteria and fungi in bee pollen and bee bread and the role that fungi play in pollen storage.In order to explore the diversity of fungi and bacteria in bee pollen and bee bread in Apis cerana and Apis mellifera,which have the largest quantity of bee pollen in China,the species and enzyme production capacity of the fungi in bee pollen,bee bread and bee gut were studied by culturable method.In order to better understand the microbial diversity in bee pollen and bee bread,high-throughput sequencing technology was used to further reveal the dynamic changes of bacteria and fungi in bee pollen and bee bread.The results are as follows:Firstly,the fungi and bacteria in the two kinds of bee pollen and bee bread were screened and cultured,and their related enzyme production ability was tested.The results showed that the flower sources of the two kinds of bees were obviously different,and after 48-72 h microbes the p H values of bee pollen and bee bread of A.mellifera and A.mellifera were significantly different.Screening of 37 filamentous fungal strains and 9 bacteria strains were isolated from the samples,the most common strains of genus is Trichoderma,Penicillium,and Mucor.Among the 37 filamentary fungial strains,32 produced organic acids,and 17 grew faster than Chalkbrood.Four fungal isolates inhibited Chalkbrood,and few strains could release protease and lipase.The filamentous fungi may encourage bees to store pollen by releasing organic acids.In addition,some filamentous fungi have shown the potential to inhibit symbiotic/contaminating bacteria and pathogens.At the same time,the bee gut fungal species of A.mellifera were also investigated.43 filamentous fungi were isolated by culture,most of which belonged to Alternaria,Penicillium,Aspergillus and Cladosporium.Among them,19 fungi strains produced lipase,26 fungi strains produced protease,and 13 fungal strains had competition with Chalkbrood.Aspergillus and Talaromyces showed strong competition.The three fungal strains could inhibit the growth of Chalkbrood,and Aspergillus could significantly inhibit the growth of Chalkbrood.Secondly,the results of the study on the fungal diversity of bee pollen and bee bread(A.cerana and A.mellifera)showed that the average number of fungal OTUs in the two kinds of bee bread was lower than that of bee pollen,but the difference was not significant,and there were also significant differences between the two species(Simpson P < 0.01).At the phylum level,Ascomycota was the most important fungal phylum,accounting for 93.55%.Basidiomycota was the second,accounting for only 5.65%.At the genus level,Cladosporium,a dominant fungus,accounts for 15-75% and 52.20% respectively.From bee pollen to bee bread,Cladosporium remained the dominant fungus,accounting for a relatively large proportion.In addition,there was no significant difference in the proportion of Cladosporium in bee pollen between the two kinds of bees.Linear discriminant analysis(Lefse)and one-way ANOVA showed that the abundance of several microbial groups was affected by pollen type,but not by bee species.Penicillium(LDA = 4.6795;P < 0.001),Aspergillus(LDA = 4.5089;P < 0.001),and the genus Alternaria(LDA = 4.6169;P = 0.048)were affected by bee pollen,only Cladosporium(LDA = 5.9206;P = 0.32)affected by bee bread.Finally,high-throughput sequencing technology was used to analyze the 16 S r RNA gene sequences of different types of bee pollen and bee bread microorganisms in A.cerana and A.mellifera to compare the species composition and abundance information of the microbiota.At the phylum level,Proteobacteria accounted for 92.12%,followed by Firmicutes(6.66%)and the other five phylum(less than 1%).At the genus level,the genus Escherichia-shiga accounts for more than 15% to 50% of the total species of bee pollen in the 12 samples of two kinds of bees.The genus Parococcus has a high content in A.mellifera and a low content in A.cerana.In the pollen of A.cerana,the dominant microbiota was Rosenbergeilla and Buttiauxella,while the content of the A.mellifera pollen was less(P = 0.019;unequal variance ttest).After 48-72 hours of fermentation,bee pollen was transformed into bee bread,and the total bacterial content ratio of bee bread and bee pollen changed.The proportion of Escherichia-Shiga in all samples dropped sharply to less than 10%(P < 0.001;t-test).In this study,the core gut microbiota of bees was found in both the bee pollen and the bee bread.The core gut microbiota may not come from the pollen material,but from the social interaction between peers in the bee nest.