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. Ptaszyńska,
Przemyslaw Latoch,
Paul J. Hurd,
Andrew Polaszek,
Joanna Michalska-Madej,
Łukasz Grochowalski,
Dominik Strapagiel,
Sebastian Gnat,
Daniel Załuski,
Marek Gancarz,
Robert Rusinek,
Patcharin Krutmuang,
Raquel Martín Hernández,
Mariano Higes Pascual,
Agata L. Starosta
Affiliations
Aneta A. Ptaszyńska
Department of Immunobiology, Faculty of Biology and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19 Str., 20-033 Lublin, Poland
Przemyslaw Latoch
Polish-Japanese Academy of Information Technology, Koszykowa 86 Str., 02-008 Warsaw, Poland
Paul J. Hurd
School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK
Andrew Polaszek
Department of Life Sciences, Insects Division, Natural History Museum, London SW7 5BD, UK
Joanna Michalska-Madej
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland
Łukasz Grochowalski
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland
Dominik Strapagiel
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, 90-231 Łódź, Poland
Sebastian Gnat
Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Preclinical Veterinary Sciences, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
Daniel Załuski
Department of Pharmaceutical Botany and Pharmacognosy, Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Marie Curie-Skłodowska 9, 85-094 Bydgoszcz, Poland
Marek Gancarz
Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4 Str., 20-290 Lublin, Poland
Robert Rusinek
Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4 Str., 20-290 Lublin, Poland
Patcharin Krutmuang
Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
Raquel Martín Hernández
Centro de Investigación Apícola y Agroambiental (CIAPA), Laboratorio de Patología Apícola, IRIAF Instituto Regional de Investigación y Desarrollo Agroalimentario y Forestal, Consejería de Agricultura de la Junta de Comunidades de Castilla-La Mancha, Camino de San Martín s/n, 19180 Marchamalo, Spain
Mariano Higes Pascual
Centro de Investigación Apícola y Agroambiental (CIAPA), Laboratorio de Patología Apícola, IRIAF Instituto Regional de Investigación y Desarrollo Agroalimentario y Forestal, Consejería de Agricultura de la Junta de Comunidades de Castilla-La Mancha, Camino de San Martín s/n, 19180 Marchamalo, Spain
Agata L. Starosta
Laboratory of Gene Expression, ECOTECH-Complex, Maria Curie-Sklodowska University, ul. Gleboka 39, 20-612 Lublin, Poland
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: 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.
