**1. Introduction**

Next-generation sequencing (NGS) is a culture-independent method often used for studying entire microbial communities, and helps to understand how microbes influence health and diseases of humans and animals including the honeybee [1–3]. Adult honeybees harbour a specialized gut microbiota of relatively low complexity with diet as a major factor influencing differences in bacterial loads [4]. Although honeybee microbiome core species composition is quite consistent regardless of environmental, geographical and genetic differences between specimens [2], some studies indicate that it can be sensitive to infection, changes in diet, malnutrition and many anthropogenic activities, such as extensive pesticide use and urban land-use changes [5–7].

Species within the *Apis* genus share fewer than 10 core species members, including *Lactobacillus*, *Bifidobacterium*, *Neisseria*, *Pasteurella*, *Gluconobacter*, *Snodgrassella* and *Gilliamella* [8–11]. Bacteria present in the honeybee gut provide numerous beneficial effects: they help digest and absorb necessary compounds and microelements, protect against mild poisonings with xenobiotics, acidify their environment which protects the gut from pathogenic microbes, e.g., *Paenibacillus larvae* that causes foulbrood, or *N. ceranae* that causes nosemosis [12,13]. They also have immunomodulation effects improving bees' immunity, strengthening the condition of the colony and prolonging bees' lives [11]. From the kingdom Fungi, yeasts are prevalent organisms in every environment in which bees conduct their life cycle, and can be isolated, for example, from honey and nectar. Honey microflora is composed of Gram-positive bacteria and yeasts, such as *Saccharomyces rouxi, S. mellis*, *S. bisporus*, *S. roesi*, *S. bailli*, *S. heterogenicus*, *Pichia (Hansenula) anomala*. The pollen reserves flora, which is dominated by bacteria from the genera *Pseudomonas* and *Lactobacillus*, and fungi from the genera Saccharomyces, Candida and Cryptococcus, far outnumbers the microflora of the honey [14–18]. Surprisingly, the gut flora of healthy, free-flying bees contains only a few yeasts if any, and diseases, malnutrition, antibiotics and insecticides cause an increase in the number of yeasts [16,19,20]. Therefore, an increased number of yeast colonies isolated from bees' guts may be considered as a stress indicator. However, recent work of Tauber et al. [5,21] suggested that, in general, yeasts are important during a younger bee's life, which includes in-house duties to feed the hive, and that the yeast community becomes less essential to the honeybee after foraging begins. Honeybee colonies are

complex super-organisms where social immune defences, natural homeostatic mechanisms, microbiome diversity and function play a major role in disease resistance. However, there is still little known about connections between, and variation among bee pathogens, bee microbiota and anthropogenic changes of environment.

Currently, in developed countries, anthropogenic landscapes are the most impacting features and include those created either directly by human activity, or indirectly by natural processes triggered by human activity [22–24]. Not only has human activity influenced geological features, but it has also considerably affected flora and fauna [25]. The loss in biodiversity is often described as the sixth mass extinction, and a slump in insect mass and biodiversity is so spectacular that the term Insectaggedon has been used to describe the phenomenon [26–30].

Recent research shows insects to be dying out eight times faster than mammals, birds, or reptiles [26,31,32]. Most noteworthy factors behind the decline of insects are inappropriate application of pesticides, increased use of fertilizers and intense agronomic activities, highly intensive farming, insect malnutrition caused by farmland monocultures, parasites, long-term drought, long-term lack of sun, especially accompanied by low temperatures, as well as viral, bacterial, and fungal diseases [24,33]. Currently special concern is being paid to the decline of pollinators, largely because of their essential ecosystem services [20]. Therefore, both for educational purposes and as a way to preserve pollinator populations many urban pollinator initiatives have arisen recently (e.g.,"Life + Urbanbees" [34], "City Bees" [35], "Urban Beekeeping" [36]). Urban colonies were shown to be more productive than rural ones, as they had access to a greater number and variety of plant species, allowing honeybees to diversify nectar sources and produce honey at a higher rate [37,38]. On the other hand, wild pollinators such as *Bombus* and *Lasioglossum* spp. were negatively affected by urbanization, but increasing the abundance and richness of floral resources could partially compensate this effect [39]. Effects of urbanization on bees are complex, variable and not well-understood [40,41]. Therefore, the aim of this study was to use the amplicon sequencing of variable 16S rRNA and ITS2 to screen honeybee colonies originating from different urban areas, and to check if "the bees really love the city".

#### **2. Results and Discussion**

Adult honeybees harbour specialized gut microbiota of relatively low complexity with five core bacterial strains [3,4,9,42,43]: *Lactobacillus* Firm-4 and Firm-5 (Firmicutes), *Giliamella* (γ-proteobacteria, Orbales), *Snodgrassella* (γ-proteobacteria, Neisseriaceae), *Bifidobacterium* (Actinobacteria) and a number of elective bacterial strains, including *Frischella* (γ-proteobacteria, Orbales), *Bartonella* (α-proteobacteria, Rhizobiales), *Commensalibacter* (αproteobacteria, Acetobacterales) and *Bombella* (α-proteobacteria, Acetobacterales), which was also confirmed in this study (Figures 1 and 2, Supplementary Materials Tables S1 and S2).

NGS was used successfully for taxonomic assessment of pollen and plants from many ecological and palynological studies, and to determine plant–pollinator interactions, or to confirm the floral composition of honey [3,44–47].

**Figure 1.** Composition of bacteria (**A**), fungi (**B**) and pollen (**C**) from Polish honeybee samples.

**Figure 2.** Composition of bacteria (**A**), fungi (**B**) and pollen (**C**) from UK, Greek, Spanish, and Thai bee samples.
