**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**

**Aneta A. Ptaszy ´nska 1,2,\*,†, Przemyslaw Latoch 3,4, Paul J. Hurd 2, Andrew Polaszek 5, Joanna Michalska-Madej 6, Łukasz Grochowalski 6, Dominik Strapagiel 6, Sebastian Gnat 7, Daniel Załuski 8, Marek Gancarz 9,10, Robert Rusinek 9, Patcharin Krutmuang 11,12, Raquel Martín Hernández 13,14, Mariano Higes Pascual <sup>13</sup> and Agata L. Starosta 4,15**


**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

**Citation:** Ptaszy ´nska, A.A.; Latoch, P.; Hurd, P.J.; Polaszek, A.; Michalska-Madej, J.; Grochowalski, Ł.; Strapagiel, D.; Gnat, S.; Załuski, D.; Gancarz, M.; et al. 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. *Pathogens* **2021**, *10*, 381. https://doi.org/ 10.3390/pathogens10030381

Academic Editor: Giovanni Cilia

Received: 22 January 2021 Accepted: 18 March 2021 Published: 22 March 2021

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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: *Orbales*, *Gilliamella*, *Snodgrassella*, 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.

**Keywords:** *Apis mellifera*; 16S rRNA; ITR2; NGS; *Nosema apis*; *Nosema ceranae*; *Nosema bombi*; *Acarapis woodi*; Trypanosomatida; *Crithidia* spp.; neogregarines; *Apicystis* spp.; antropocene; insectageddon; urban area; urban environment; bee biology
