Influence of external and internal environmental factors on intestinal microbiota of wild and domestic of animals factors on intestinal microbiota Influence of external and internal environmental Influence of external and internal environmental factors on intestinal microbiota of wild and domestic animals A. Umanets Alexander Umanets Propositions 1. Intestinal microbiota and resistome composition of wild animals are mostly shaped by the animals’ diet and lifestyle. (this thesis) 2. When other environmental factors are controlled, genetics of the host lead to species- or breed specific microbiota patterns. (this thesis) 3. Identifying the response of microbial communities to factors that only have a minor contribution to overall microbiota variation faces the same problems as the discovery of exoplanets. 4. Observational studies in microbial ecology using cultivation- independent methods should be considered only as a guide for further investigations that employ controlled experimental conditions and mechanistic studies of cause-effect relationships. 5. Public fear of genetic engineering and artificial intelligence is not helped by insufficient public education and misleading images created through mass- and social media. 6. Principles of positive (Darwinian) and negative selection govern the repertoire of techniques used within martial arts. Propositions belonging to the thesis, entitled Influence of external and internal environmental factors on intestinal microbiota of wild and domestic animals Alexander Umanets Wageningen, 17 October 2019 Influence of external and internal environmental factors on intestinal microbiota of wild and domestic animals Alexander Umanets Thesis committee Promotor Prof. Dr H. Smidt Personal chair at the Laboratory of Microbiology Wageningen University & Research Other members Prof. Dr Michiel Kleerebezem, Wageningen University & Research Dr John Penders, Maastricht University Dr Leo Lahti, University of Turku, Finland Dr Nadia Everaert, University of Liège, Belgium This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences) Influence of external and internal environmental factors on intestinal microbiota of wild and domestic animals Alexander Umanets Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 17 October 2019 at 11 a.m. in the Aula. Alexander Umanets Influence of external and internal environmental factors on intestinal microbiota of wild and domestic animals, 208 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2019) With references, with summary in English. ISBN: 978-94-6395-093-0 DOI: https://doi.org/10.18174/498870 Table of contents Chapter 1 General introduction and thesis outline..................................... 7 Chapter 2 Occupancy strongly influences faecal microbial composition of wild lemurs................................................................................... 23 Chapter 3 Effects of seasonality and previous logging on faecal helminth-microbiota associations in wild lemurs...................... 57 Chapter 4 Variation of caecal microbial composition among performance-tested Italian Landrace, Italian Large White, and Italian Duroc pig breeds demonstrate the effect of host genetic background on microbiota diversity............................... 89 Chapter 5 Differences in intestinal microbiota composition between White-toothed Shrews and Wood Mice shapes differences in their resistome composition......................................................... 117 Chapter 6 General discussion and future perspectives............................... 141 Appendices References .................................................................................. 162 Summary...................................................................................... 197 Chapter 1 General introduction and thesis outline They were the first Microbes. They have been around almost since the beginning of times on Earth - the first life form appeared on the face of our planet around 3.45 billion years ago [1], and only a billion years after planet Earth was formed from a hot cloud of gasses and space dust [2]. Much later, around 600 million years ago in the Late Precambrian period, the first multicellular organisms appeared [3], which means that single-celled organisms had seven times more time to evolve and adapt, and they didn’t waste it. A tree of life published in 2016 by Laura A. Hug et al. [4] depicted 92 bacterial and 26 archaeal phyla, providing a good visual representation of the tremendous diversity of prokaryotic life. Phylogenetic diversity of prokaryotes goes hand to hand with their ability to colonise the widest range of natural and man-made environments and to utilise various chemical compounds to recover energy. Microbial life can be found practically everywhere: in hot springs near volcanoes [5], the Arctic sea [6], outside and inside of human and animal bodies [7], and even possibly on other planets [8]. Since its origin, eukaryotic and later also multicellular life has never been isolated from its prokaryotic ancestors and relatives. The life on Earth became more complex with time, including intricate relationships between all its domains. Looking at the modern time eukaryotic-prokaryotic relationships we can find fascinating examples where the entire life of an animal depends on a symbiosis with microorganisms. It is known that more than 10% of insects rely heavily in their metabolism on obligate bacterial symbionts [9]. A textbook example is the presence of a special type of cells (bacteriocytes) in aphids that host an obligatory bacterial symbiont, Buchnera aphidicola, that covers the aphid’s needs in amino acids in exchange for shelter and sugar [10]. In the sea bacteria-host mutualism can go into extremes, as exemplified by some oligochaetes such as Olavius crassitunicatus that completely lost (or never developed) a digestive system and rely for the acquisition of nutrients completely on microbial communities residing within their body that produce organic nutrients via chemosynthesis [11]. When we are talking about mammals we will 8 8 not find suh eoti eaples of bateria-host utualis howeer the iportane of irobes in their life and wellbein an hardly be oerestiated. Chapter 1 Chapter Microbes and humanity een huanity has been usin iroorganiss unintentionally for their own ood as lon as iiliation eists. ne of the first doestiated irooraniss were probably yeasts that were used for beer brewin and bread ain as early as 10000 years ao followed by Streptococcus and Lactobacillus speies that ae to play an iportant role toether with doestiation of il produin anials and the disoery of saouriness and health benefits of ferented il produts 12. Appliation of irooraniss beae ore and ore ople alonside with tehnoloial proress, allowin huanity to tale hallenes that were not feasible before suh as prodution of aluable heial opounds and pharaeutials usin enetially odified irooraniss 1. oweer until only a few years ba irobes were pereied alost elusiely as soethin inherently harful when huan and anial health were onsidered. he neatie reputation of bateria is not surprisin and started with the postulation of irobial aetioloy of diseases by Louis asteur and obert oh 1. noffiially a war on irobes had been delared. Only in recent years a notion about “beneficial microorganisms” entered the ass edia and has eer sine been a hot topi. oweer ore than a hundred years ao lya ehnio was already onined that ertain lati-aid bateria ould be a reason for huan loneity, and he was atiely worin on a theory about the interation between host and intestinal irooraniss 1 16. A few years later Alfred issle isolated the first well defined and probably the ost widely used probioti strain Escherichia coli Nissle 11 with remarable antaonisti properties aainst Salmonella 1. We went a lon way fro the disoeries of lya ehnio and Alfred issle and the odern ae of hih- throuhput tehnoloies and bi data allowed us to tae a loo not only at indiidual ebers of irobial ounities but also onsider irobial 9 9 consortia as a ole n articular bacteria delling along te gastrointestinal (G tract of umans and animals attracted a lot of attention and soed to be a fascinating topic of research. Currently the term “microbiota” is used to depict tese and oter microbial communities, but also the term “microbiome” is often used to describe microbial comosition function and enironmental factors togeter e origin of te term microbiome itself is not clear and often osua ederberg is credited as a erson o used it first in oeer te term “microbiota” has been used since 1960 for handling of germ-free animals and in 198 te book “Mycoparasitism and plant disease control” provided a definition [18]. “Characteristic microbial community occupying a reasonably well defined abitat wic as distinct ysio-chemical properties” was that first definition gien to microbiome [19] t is clear tat originally te term microbiome as defined as a microbial community liing in any ecological system ic does not ae to be te uman body ntil no e can see tat a uman centric definition is idely used. For eamle te erriam-ebster dictionary maes an emasis on “microorganisms living in or on the human body” in an article on te ord “microbiota”