~ FACULTY 1/:0., FACULTY OF (Ç; OF SCIENCES ~ BIOSCIENCE ENGINEERING The gut microbiota of bumblebees: a treasure chest of biodiversity and functionality Jessy Praet Supervisors: Prof. dr. Peter Vandamme Prof. dr. ir. Guy Smagghe Dissertation submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) of Science: Biochemistry and Biotechnology (Ghent University) Laboratory of Microbiology (LM{UGent) ............... Laboratory of Agrozoology 1111111 GHIENT UNIVERSITY Praet, J. (2017). The gut microbiota of bumblebees: a treasure chest of biodiversity and functionality. Ph.D. thesis, Ghent University, Belgium. Copyright © 2017 Jessy Praet Printed by University Press, Zelzate, Belgium jhttp://www.universitypress.be ISBN-number: 978-94-6197-544-7 All rights reserved. No part of this thesis protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage or retrieval system without written permission of the author and supervisors. Cover design by Laurien Troch This work was funded by the Special Research Council of Ghent University. Ph.D. thesis, Faculty of Sciences, Ghent University, Belgium. Publicly defended in Ghent, Belgium, on June 29, 2017. Examination Committee Prof. Dr. Savvas SAVVIDES (Chairman) Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE) Faculty of Sciences, Ghent University, Ghent, Belgium VIB Inflammation Research Center VIB, Ghent, Belgium Prof. Dr. Anne WILLEMS (Secretary) Laboratory of Microbiology (LM-UGent) Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Peter VANDAMME (Supervisor) Laboratory of Microbiology (LM-UGent) Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Ir. Guy SMAGGHE (Supervisor) Laboratory of Agrozoology Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Prof. Dr. Alejandra VASQUEZ Medical Microbiology Faculty of Medicine, Lund University, Lund, Sweden Prof. Dr. Ir. Nico BOON Center for Microbial Ecology and Technology Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Prof. Dr. Dirk DE GRAAF Laboratory of Molecular Entomology and Bee Pathology Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Kurt HOUF Department of Veterinary Public Health and Food Safety Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium Dr. Ivan MEEUS Laboratory of Agrozoology Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium iii English summary Bumblebees are efficient generalist pollinators and are important for food production and ecosystem integrity. Commercial bumblebee colonies are used worldwide to enhance the pollination of agricultural crops. Because of the high density and potentially higher pathogen susceptibility of commercial bees, these bees can become reservoirs with high pathogen prevalence of which pathogens can spill back to wild bumblebees. Several studies indicate that the bumblebee gut microbiota contributes to its hosts' health and that it consists of several novel species of bee specific taxa with unknown functionality. The implementation of probiotics may therefore benefit commercial bumblebee health and increase their resistance against pathogens which in turn may reduce pathogen spillover. The diversity of the bumblebee gut microbiota has mainly been studied by cultivation{ independent approaches but these studies lack taxonomic resolution. In addition, the availability of microbial isolates facilitates the analysis of their functional potential. Hence, a first goal was to properly and thoroughly inventorise the cultivable bumblebee gut microbiota through an extensive isolation campaign, coupled to state of the art species-level identification of isolates. Microbial isolates were obtained from the guts of 60 bumblebees belonging to four Bombus species by using a rich medium (AC agar) in combination with selective media for the isolation of LAB (MRS agar) and bifidobacteria (MTPY and M144 agar) at 37°C and different atmospheres. By using these isolation conditions and the high-throughput MALDI-TOF MS tool for dereplication, a set of 1940 isolates were obtained belonging to 52 species. We demonstrate that the bacterial species diversity of the bumblebee gut as suggested by 16S rRNA amplicon studies is considerably underestimated as several phylotypes are represented by multiple species and several novel species were isolated for the first time in this thesis. We also show that specific yeast species can be highly prevalent in the bumblebee gut and a proper evaluation of their diversity and functional potential is mandatory. While representatives of most phylotypes reported earlier and detected in the present study were isolated, the isolation of two low abundant but frequently detected phylotypes i.e. Gamma-II and Lacto-4, remained elusive. In accordance with previously reported studies, we found significant differences between the microbial community profiles of different bumblebee species which might be explained by a different host ecology or physiology. v English summary A second goal was to explore the functional potential of the isolates through pathogen inhibition assays and a pectin degradation assay. The parasite Crithidia bombi is highly prevalent in bumblebees and is associated with reduced ovary growth and slow colony growth. A 96-well inhibition assay was developed and implemented to test if supernatant of the isolates can inhibit growth of C. bombi. Six isolates were obtained with in vitro activity against C. bombi. The active compound of five Weissella isolates is likely hydrogen peroxide while that of a Streptomyces albidoflavus isolate remains elusive and is probably non-proteinaceous. Honeybees suffer from severe overwintering declines which are caused by a combination of factors like pesticide use and pathogens. As the honeybee gut microbiota is similar to that of bumblebees, microbial isolates from the bumblebee gut with activity against honeybee pathogens might also be good candidates for honeybee probiotics. Therefore, a well diffusion assay was implemented to investigate if the isolates are able to inhibit growth of the honeybee pathogens Paenibacillus larvae and Melissococcus plutonius and the bee pathogen Ascosphaera apis. Several isolates were obtained with the capacity to inhibit growth of these pathogens in vitro but the mechanism of action remains speculative. Pectin is a compound of the pollen wall which is toxic to bees and degradation of pectin by gut micro-organisms may enhance the release of undigested nutrients and reduce pectin-mediated toxicity. Only one isolate was obtained which degraded pectin in vitro. The bumblebee gut microbiota consists of several novel species. A third goal was therefore to formally describe and name these novel species by performing the required polyphasic taxonomic studies and by making reference cultures of these novel species available to the scientific world through their deposit in public culture collections. In the present thesis we described novel species belonging to the genera Bombella, Gilliamella, Lactobacillus, Weissella, Convivina, Bifidobacterium and Apibacter. Of these novel species Bombella intestini, four novel Gilliamella species, Lactobacillus bombicola, Bifidobacterium commune and Apibacter mensalis belong to bacterial phylotypes previously detected in the bumblebee gut. vi Nederlandstalige samenvatting Hommels zijn effici¨ente bestuivers van verschillende plantenfamilies en zijn belangrijk voor de voedselproductie en het behoud van ecosysteem integriteit. Commerci¨elehommelkolonies worden wereldwijd ingezet om de bestuiving van landbouwgewassen te bevorderen. Door de hoge densiteit aan commerci¨elekolonies en de mogelijks hogere vatbaarheid van commerci¨ele hommels kunnen deze hommels een reservoir vormen met een hoge pathogeen prevalentie waarvan pathogenen kunnen overgedragen worden naar wilde hommels. Verschillende studies geven aan dat de darmmicrobiota van hommels bijdraagt tot hun gezondheid en dat deze microbiota bestaat uit verschillende nieuwe soorten van bij-specifieke taxa met een ongekende functionaliteit. Het gebruik van probiotica zou de resistentie van commerci¨elehommels tegen pathogenen kunnen verhogen wat op zijn beurt de overdracht van pathogenen kan verminderen. De diversiteit van de darmmicrobiota van hommels werd voornamelijk bestudeerd door middel van cultivatie-onafhankelijke benaderingen waarvan de taxonomische resolutie beperkt is. Daarenboven vergemakkelijkt de beschikbaarheid van microbi¨eleisolaten het onderzoek naar hun functionele potentieel. Een eerste doel van deze thesis was daarom om een grondig inventaris te maken van de kweekbare micro-organismen in de hommeldarm door middel van een uitgebreide isolatiecampagne gekoppeld aan state of the art identificatie van de isolaten op soortniveau. Microbi¨eleisolaten werden verkregen uit de darmen van 60 hommels behorende tot vier hommelsoorten door het gebruik van een rijk medium (AC agar) in combinatie met selectieve media voor de isolatie van melkzuurbacteri¨en (MRS agar) en bifidobacteri¨en(MTPY en M144 agar) bij 37°C en verschillende atmosferen. Door het gebruik van deze isolatiecondities en de MALDI-TOF MS dereplicatie methode met een hoge doorvoercapaciteit werd een set van 1940 isolaten bekomen die 52 soorten omvatten. We tonen aan dat de bacteri¨elesoortendiversiteit van de hommeldarm zoals gesuggereerd door 16S rRNA amplicon studies aanzienlijk onderschat is aangezien meerdere soorten