Amplicon Sequencing of Variable 16S rRNA from Bacteria and ITS2 Regions from Fungi and Plants, Reveals Honeybee Susceptibility to Diseases Results from Their Forage Availability under Anthropogenic Landscapes

Abstract

European Apis mellifera and Asian Apis cerana honeybees are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of the 16S rRNA from bacteria and the internally transcribed spacer (ITS) regions from fungi and plants allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollen collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees collected at various time points from anthropogenic landscapes such as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial content of honeybee intestine as well as fungi and pollens. Furthermore, isolated DNA was used as the template for screening pathogens: Nosema apis, N. ceranae, N. bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi). We conclude that differences between samples were mainly influenced by the bacteria, plant pollen and fungi, respectively. Moreover, honeybees feeding on a sugar based diet were more prone to fungal pathogens (Nosema ceranae) and neogregarines. In most samples Nosema sp. and neogregarines parasitized the host bee at the same time. A higher load of fungi, and bacteria groups such as Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and other unidentified bacteria was observed for Nosema ceranae and neogregarine infected honeybees. Healthy honeybees had a higher load of plant pollen, and bacteria groups such as: γ-proteobacteria, Orbales, Gilliamella, Snodgrassella, γ-proteobacteria and Enterobacteriaceae. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations, and hence pathogen attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.

A Snapshot Picture of the Fungal Composition of Bee Bread in Four Locations in Bulgaria, Differing in Anthropogenic Influence

“…However, some confusion could arise as a result of the relatively high percentages of unclassified tags which varied approximately between 94% and 98%. A high percentage of unclassified fungi when ITS-based methods are employed is not unusual and could be explained by the incompleteness of the international databases with data of environmental samples [8,19,40]. For example, it varies from above 50% to 80% for environmental samples [40][41][42] to "significant" [43] and above 98% [44] for fermented foods and products.…”

“…Because the microbial composition of the honeybees' microbiomes, as well as those of the honeybee products, is strongly influenced by the landscape and anthropogenic pressure [19], we decided to investigate the fungal composition in bee bread at the end of June 2020 in four different locations in Bulgaria (Figure 1). This period was chosen as one of the most active concerning the honeybees' foraging activities while the locations were selected because of the different degrees of anthropogenic pressure, as well as the landscape and the climatic features.…”

Overwintering honeybees maintained dynamic and stable intestinal bacteria

“…Alteration of gut microbiota influences host metabolism and may affect the storage of energy in honeybees. Some studies had suggested that changes in diversity of gut microbiota are associated with some diseases and opportunistic bacteria 43 , included fungal pathogens and neogregarines 21 . This would benefit honeybees to reduce parasite and pathogenic infection for successful overwintering 24 .…”

“…During the growth stage of honeybees, the intestinal bacteria are also involved in growth improvement and immunomodulation of its hosts 15 , 19 . The intestinal bacteria of a healthy honeybees are characterized by its dynamic stability 21 . However, it can show dysbiosis or disequilibrium when challenged by many factors alone or combination, such as a different diet 22 , pesticide exposure 23 , parasite and pathogen infection 24 , and behavioral tasks 25 .…”

A Meta-Analysis of the 16S-rRNA Gut Microbiome Data in Honeybees (Apis Mellifera)

“…Finally, since this is an active research area, several studies (such as Ptaszyńska et al, 2021 64 ) were added throughout our analysis that would have fulfilled inclusion criteria. This underlines the importance of a curated and active database as proposed by Engel et al We intend to include all studies published since the initial search in a later iteration of this study.…”