sign in sign up
<<
Home , Eugene Rosenberg, Martinus Beijerinck

The of Humans, Animals, , and the Environment 2

Eugene Rosenberg Microbiomes Current Knowledge and Unanswered Questions The Microbiomes of Humans, Animals, Plants, and the Environment

Volume 2 This series covers topics from all natural habitats. Microbiome research is a vibrant field of science that offers a new perspective on with a more comprehensive view on different () living and working together as a community (microbiome). Even though microbial communities in the environment have long been examined, this scientific movement also follows the increasing interest in microbiomes from humans, animals and plants. First and foremost, microbiome research tries to unravel how individual species within the community influence and communicate with each other. Addi- tionally, scientists explore the delicate relationship between a microbiome and its habitat, as small changes in either, can have a profound impact on the other. With individual research volumes, this series reflects the vast diversity of Microbiomes and highlights the impact of this field in Microbiology.

More information about this series at http://www.springer.com/series/16462 Eugene Rosenberg

Microbiomes Current Knowledge and Unanswered Questions Eugene Rosenberg Department of Molecular Microbiology & Tel Aviv University Givat Shmuel, Tel Aviv, Israel

ISSN 2662-611X ISSN 2662-6128 (electronic) The Microbiomes of Humans, Animals, Plants, and the Environment ISBN 978-3-030-65316-3 ISBN 978-3-030-65317-0 (eBook) https://doi.org/10.1007/978-3-030-65317-0

# Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland To Ilana, my scientific and life partner Preface

The great scientific news that greeted this century was the campaign to decode the human genome. We must now remind ourselves that much of the biological composition of our bodies consists of genomes other than the human. Multitudes of and occupy our skin, our mucous membranes, and our intestinal tract. They are likely to play a much larger role in developing—and resisting—disease than we realize. Understanding this cohabitation of genomes within the human body—what I call the microbiome—is central to understanding the dynamics of health and disease. Joshua Lederberg (2004)

We are in the midst of a paradigm change in . Plants and animals, including humans, can no longer be considered individuals, but rather, all are consisting of complex interactions between the host and abundant and diverse symbiotic microorganisms. During the last two decades, numerous studies have demonstrated that these symbionts play a critical role in many functions of macroorganisms, including metabolism, behavior, development, adaptation, and evolution (Gilbert et al. 2012; McFall-Ngai et al. 2013). Thus, individual phenotypes result from the combined expression of the host and microbiome genomes, leading to the popularization of notions of the and the hologenome (Zilber- Rosenberg and Rosenberg 2008; Rosenberg and Zilber-Rosenberg 2013, 2018). I would like to mention briefly how Ilana Zilber-Rosenberg and I came upon the hologenome concept. In 1996, we discovered bacterial bleaching of (Kushmaro et al. 1996, 1997). After six years of studying the mechanisms of infection (reviewed in Rosenberg and Falkovitz 2004), we observed that the had become resistant to infection and bleaching by the specific coral , Vibrio shiloi. Because corals possess a restricted adaptive immune system and do not produce antibodies, we presented the coral hypothesis (Reshef et al. 2006) to explain the coral development of resistance to infection by V. shiloi. The hypothesis posits that the corals acquired “beneficial” bacteria from the marine environment that prevented infection by the pathogen. If it is possible to have epidemics of , why is it not possible (even more likely) to have epidemics of beneficial bacteria? They simply generally go unnoticed. Subsequently, we published data that support the coral probiotic hypothesis (Mills et al. 2013). A dynamic relationship exists between symbiotic microorganisms and corals under different environmental conditions that selects for the most advantageous coral holobiont in the context of the prevailing conditions. vii viii Preface

Although the coral probiotic hypothesis inspired the hologenome concept, the concept was developed by consideration of the vast amount of data published by others. I especially acknowledge Forest Rohwer, whose pioneer article on the coral holobiont had a strong influence on us (Rohwer et al. 2001), Jan Sapp, whose book, Evolution by Association: A History of , was a stimulating introduction into the subject (Sapp 1994), and Eva Jablonka and Marion Lamb for their stimulating book, Evolution in Four Dimensions, which argues that there is more to heredity than genes (Jablonka and Lamb 2005). In a previous book (Rosenberg and Zilber-Rosenberg 2013), we began by putting forth the hologenome concept of evolution. This was followed by a systematic review of the experimental evidence that existed at the time supporting the concept. This book takes a different approach. I first describe in some detail current knowl- edge of the properties of microbiomes, including their microbial abundance and diversity (Chap. 2), interaction with their hosts (Chap. 4), modes of transmission (Chap. 5), and role in genetic variation (Chap. 8) and evolution (Chap. 9)of holobionts. Chaps. 6 and 7 discuss eukaryotic microbiota and viruses, respectively. After presenting information on microbiomes, I then propose that the hologenome concept of evolution provides a useful framework for understanding these data, taking into consideration published theoretical arguments, supporting (e.g., Fraune and Bosch 2010; Bordenstein and Theis 2015; Roughgarden et al. 2018) and challenging (e.g., Moran and Sloan 2015; Douglas and Werren 2016) the concept. Chap. 10 discusses microbiomes in medicine and , including , prebiotics, synbiotics, fecal transplantation, and phage therapy. I conclude with a chapter on some philosophical and sociological implications of microbiome research. One of the problems I faced in completing this book was each time I completed a draft, important new publications appeared, which caused me to rewrite many of the chapters. This was not surprising because of the fast-moving nature of this subject (see Fig. P.1). In addition to many high impact journals now devoting sections to microbiomes, several relatively new journals are devoted entirely to microbiome research, including Animal Microbiomes, Microbiome, , Cell Host and Microbiome, mBio, mSystems, and Gut Microbes. The literature search was completed in July 2020. I thank my partner, Ilana Zilber-Rosenberg, for the enormous help she gave me in writing this book, providing me current references, discussing every chapter, and moderating my tendency to overstate conclusions from the existing data. I also thank Ed Kosower, Gil Sharon, Ariel Kushmaro, Ehud Lamm, Omry Koren, and David Gutnick for providing useful references and interesting discussions. It was a pleasure to work with Markus Spaeth, Bibhuti Sharma, Andrea Schlitzberger, and the other editors of Springer- Verlag in bringing the manuscript to publication. Preface ix

Fig. P.1 Occurrence of terms “Holobiont” (N ¼ 695) and “Hologenome” (N ¼ 102) in Web of Science Core Collection from 1991 to 2018. Taken from Simon et al. (2019)

References

Bordenstein, S. R., & Theis, K. R. (2015). Host biology in light of the microbiome: Ten principles of holobionts and hologenomes. PLoS Biol, 13, e1002226. Douglas, A. E., & Werren, J. H. (2016). Holes in the hologenome: Why host- microbe symbioses are not holobionts. mBio, 7(2), e02099-15. Fraune, S., & Bosch, T. C. G. (2010). Why bacteria matter in animal development and evolution. Bioessays, 32, 571–580. Gilbert, S. F., Sapp, J., & Tauber, A. I. (2012). A symbiotic view of life: We have never been individuals. The Quarterly Review of Biology, 87, 325–341. Jablonka, E. & Marion J. Lamb, M. J. (2005). Evolution in four dimensions: Genetic, epigenetic, behavioral, and symbolic variation in the history of life. Cambridge: MIT Press. Kushmaro, A., Loya, Y., Fine, M., et al. (1996). Bacterial infection and coral bleaching. Nature, 380, 396. Kushmaro, A., Rosenberg, E., Fine, M., et al. (1997). Bleaching of the coral Oculina patagonica by Vibrio AK-1. Marine Ecology Progress Series, 147, 159–165. Lederberg, J. (2004). Of men and microbes, New Perspectives Quarterly, 21(4), 92– 96. McFall-Ngai, M., Hadfield, M. G., Bosch, T. C. G., et al. (2013). Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the National Academy of Sciences of the United States of America, 110(9), 3229– 3236. x Preface

Mills, E., Shechtman, K., Loya, Y., et al. (2013). . Bacteria appear to play important roles both causing and preventing the bleaching of the coral Oculina patagonica. Marine Ecology Progress Series, 489, 155–162. Moran, N. A. & Sloan, D. B. (2015). The hologenome concept: Helpful or hollow? PLoS Biol, 13(12), e1002311. Reshef, L., Koren, O., Loya, Y., et al. (2006). The coral probiotic hypothesis. Environmental Microbiology, 8, 2067–2073. Rohwer, F., Breitbart, M., Jara, J., et al. (2001). Diversity of bacteria associated with the Caribbean coral Montastraea franksi. Coral Reefs, 20,85–91. Rosenberg, E., & Falkovitz, L. (2004). The Vibrio shiloi / Oculina patagonica model system of coral bleaching. Ann Rev Microbiol, 58, 143–159. Rosenberg, E., & Zilber-Rosenberg, I. (2013). The hologenome concept: Human, animal and microbiota. Heidelberg: Springer. Rosenberg, E., & Zilber-Rosenberg, I. (2018). The hologenome concept of evolution-after ten years. Microbiome 6, 78. https://doi.org/10.1186/s40168- 018-0457-9. Roughgarden, J., Gilbert, S.F., Rosenberg, E., et al. (2018). Holobionts as units of selection and a model of their population dynamics and evolution. Biol Theory, 13,44–65. https://doi.org/10.1007/s13752-017-0287-1. Sapp, J. (1994). Evolution by association: A history of symbiosis. New York: Oxford University Press. Simon, J. C., Marchesi, J. R., Mougel, C., et al. (2019). Host-microbiota interactions: From holobiont theory to analysis. Microbiome, 7(1), 5, doi, 10.1186/s40168- 019-0619-4. Zilber-Rosenberg, I., & Rosenberg, E. (2008). Role of microorganisms in the evolution of animals and plants: The hologenome theory of evolution. FEMS Microbiol Rev, 32, 723–735. Contents

1 Introduction ...... 1 1.1 History ...... 3 1.2 Definitions and Concepts ...... 6 References ...... 9 2 Composition of Microbiomes ...... 15 2.1 Abundance of Microbes in Holobionts ...... 16 2.2 Diversity of Microbes in Holobionts ...... 17 2.3 Microbiota of Invertebrates (Table 2.1) ...... 18 2.4 Microbiota of Vertebrates (Table 2.2) ...... 24 2.5 Microbiota of Plants (Table 2.3) ...... 33 2.6 Critical Analysis of Bacterial Diversity Data ...... 37 2.7 Key Points ...... 39 2.8 Unanswered Questions and Future Research ...... 39 References ...... 40 3 Dynamics of Microbiomes ...... 57 3.1 Age and Developmental Stage ...... 58 3.2 Gender and Pregnancy ...... 64 3.3 Diet ...... 66 3.4 Physical Activity ...... 70 3.5 Stress ...... 71 3.6 Temperature ...... 72 3.7 Disease ...... 75 3.8 ...... 80 3.9 Other Factors ...... 81 3.10 Key Points ...... 82 3.11 Unanswered Questions and Future Research Questions ...... 82 References ...... 82 4 Holistic Fitness: Microbiomes are Part of the Holobiont’s Fitness ... 101 4.1 Protection Against Pathogens ...... 106 4.2 Protection Against Cancer and Cardiometabolic Disease ...... 110 4.3 Provision of Nutrients ...... 111 4.4 Holobiont Metabolism ...... 115

xi xii Contents

4.5 Influence on Animal and Plant Development ...... 117 4.6 Influence on Animal Behavior ...... 124 4.7 Detoxification ...... 128 4.8 Temperature Adaptation ...... 132 4.9 Microbiomes Warm the Holobiont ...... 134 4.10 Key Points ...... 135 4.11 Unanswered Questions and Future Research ...... 136 References ...... 136 5 Transmission of Hologenomes Between Generations: Mothers Matter Most ...... 161 5.1 Modes of Transmission ...... 162 5.2 Fidelity of Transmission ...... 171 5.3 Transmission of Microbiota and Social Behavior ...... 176 5.4 Transmission and the Holobiont Concept ...... 176 5.5 Key Points ...... 179 5.6 Unanswered Questions and Future Research ...... 180 References ...... 180 6 Eukaryotic Microorganisms are Part of Holobionts ...... 195 6.1 Fungi ...... 196 6.2 Algae ...... 206 6.3 ...... 209 6.4 Key Points ...... 214 References ...... 214 7 Viruses Are Part of the Holobiont’s Fitness and Evolution ...... 231 7.1 Abundance and Diversity of Viruses in Holobionts ...... 233 7.2 Transmission of Viruses ...... 237 7.3 Viruses Are Part of the Fitness and Evolution of Holobionts: Beneficial Viruses ...... 239 7.4 Key Points ...... 253 7.5 Unanswered Questions and Future Research ...... 254 References ...... 255 8 Genetic Variation in Holobionts ...... 275 8.1 Introduction: and Lamarckism ...... 276 8.2 Modes of Variation Within Holobionts ...... 279 8.3 Microbial Amplification/Reduction ...... 279 8.4 Acquisition of Novel Symbionts and the “Hygiene Hypothesis” ...... 288 8.5 Horizontal Gene Transfer ...... 292 8.6 Inheritance of Acquired Characteristics: Lamarckism Revisited ...... 297 8.7 Key Points ...... 297 8.8 Unanswered Questions and Future Research ...... 298 References ...... 298 Contents xiii

9 Evolution of Holobionts: The Hologenome Concept ...... 317 9.1 Levels of Selection in Evolution ...... 319 9.2 Random Drift and Evolution of Holobionts ...... 322 9.3 A Microbial Explanation for Cheating and the Evolution of Altruism ...... 323 9.4 Hologenomes and the Origin of Species (Speciation) ...... 326 9.5 Evolution by Cooperation ...... 330 9.6 Competition and Cooperation in the Evolution of Biological Complexity ...... 336 9.7 Key Points ...... 339 9.8 Unanswered Questions and Future Research ...... 339 References ...... 340 10 Microbiomes in Medicine and Agriculture ...... 353 10.1 Probiotics: Variation of Holobionts by Ingestion of Live Bacteria ...... 355 10.2 Prebiotics: Variation of Holobionts by Amplification of Indigenous Microbiota ...... 366 10.3 Synbiotics ...... 369 10.4 Fecal Transplantation ...... 373 10.5 Phage Therapy ...... 376 10.6 Key Points ...... 385 10.7 Unanswered Questions and Future Research Questions ...... 386 References ...... 386 11 Microbiomes: Some Philosophical and Sociological Implications ... 413 11.1 Individualism ...... 414 11.2 Collective Memory ...... 417 11.3 Biological Cooperation Versus Social Darwinism ...... 420 11.4 Microbiomes and the Philosophy of Medicine ...... 422 11.5 Teaching and Researching Biology/Microbiology ...... 425 References ...... 425 Introduction 1

Mutually beneficial relationships between microbes and animals are a pervasive feature of life on our microbe- dominated planet. — Jeffrey Gordon

Abstract

Microbiome research began with the discovery of a wide variety of bacteria in the human mouth (oral microbiome) by Leeuwenhoek in 1683. However, it was not until the end of the 19th C, with the development of pure culture techniques by , was microbiology able to grow into a true science. During most of the twentieth century, microbiological research focused on isolating microbes that were causative agents of disease and studying their transmission and mecha- nism of pathogenesis. Subsequently and not less important, these pure culture techniques were applied as model microbial systems to discover the underlining principles of and genetics. At the time, insufficient tools were available to study the ecology of complex mixtures of microbes. An important background for subsequent microbiome research was pioneer studies of symbio- sis between specific bacteria and their hosts. Only toward the end of the 20th C, the advent of techniques for PCR and sequencing DNA, coupled with bioinfor- matics for analyzing the data opened the door for studying and understanding the vast microbial richness associated with animal and plant microbiomes. We now realize that microorganisms are not only model systems for studying genetics and physiology, but are an important part of all animals and plants. Realizing this has led to a paradigm change in how we view the biological world. Understanding the microbiome literature requires a grasp of the definition of specialized terms, many of which are presented in this chapter.

# Springer Nature Switzerland AG 2021 1 E. Rosenberg, Microbiomes, The Microbiomes of Humans, Animals, Plants, and the Environment 2, https://doi.org/10.1007/978-3-030-65317-0_1 2 1 Introduction

Keywords

Microbiome · Pure cultures · Symbiosis · Taxonomy · Holobiont

Biology is undergoing a paradigm shift. Animals and plants are no longer individuals, but systems, consisting of a host and abundant and diverse microorganisms. The significance of this realization is only beginning to be appreciated. The interaction between symbiotic microbes and their hosts affects many aspects of life. In the following chapters, we will review current knowledge of how symbiotic microorganisms contribute to the anatomy, behavior, develop- ment, metabolism, physiology, genetic variation and evolution of animals and plants. To understand the complex system of interacting host and microbiome, it is necessary to combine many scientific disciplines, such as computational tools of bioinformatics and systems biology with the more classical areas, such as microbi- ology, genetics, and . Cooperation between these different specialists requires a common language. One of the aims of this book is to present information and concepts that can provide the foundation and framework for coop- erative research (Fig. 1.1).

Fig. 1.1 Drawing by Avshalom Falk. Taken from Rosenberg (2017) 1.1 History 3

1.1 History

The past, the finite greatness of the past/For what is the present, after all, but a growth out of the past. – Walt Whitman

Microbiology, like most scientific disciplines, has advanced by observations, asking interesting questions, and developing techniques to understand these observations and answer these questions. Microbiology began with the discovery by Antony van Leeuwenhoek in 1674 of protozoa in fresh water, and in 1683, of a wide variety of bacteria in the human mouth. In essence, he was observing the oral microbiome. Between 1653 and 1673, Leeuwenhoek developed the curious hobby of constructing microscopes. Although he was not the first to build a microscope, his microscopes were among the finest of that time. Leeuwenhoek patiently improved his microscopes and techniques of observation for 20 years before he reported his results in the form of letters, written in “Nether-Dutch,” to the secretary of the Royal Society of England. After translation, the Society published the letters. For details on the life and discoveries of van Leeuwenhoek, we recommend his inspiring biography written by Clifford Dobell (1932). Following van Leeuwenhoek’s initial observations of microbes, it took 200 years for microbiology to develop into a full-blown science. The liquid suspensions that Leeuwenhoek and other pioneers in bacteriology examined under the microscope contained a wide assortment of microbes of varying sizes and shapes. Was this because a particular organism could exist in various forms, or was it the result of a mixture of different organisms, each having a fixed form? To answer this question and to test the subsequent , it became necessary to develop techniques for obtaining pure cultures, a culture containing the growth of a single kind of organism, free from other organisms. Lord Lister obtained the first pure culture in 1878, using a dilution technique. A mixed culture was continually diluted until only one was present. That organism was then allowed to grow into a pure culture. In 1881, the German physician and bacteriologist Heinrich Hermann Robert Koch developed the simple and efficient “streak-on-agar medium” technique for obtaining pure cultures that is still used today (Koch 1882). The technique proved so valuable that by 1900, twenty-one microbes that caused diseases were identified. (Koch won the 1905 Nobel Prize in Physiology or Medicine.)

As soon as the right method was found, discoveries came as easily as ripe apples from a tree. — Robert Koch

At the beginning of the 20th C, two schools of microbiology emerged. The Robert Koch/ School of microbiology employed pure cultures to isolate and characterize pathogenic bacteria. The second school of microbiology, led by the Russian and ecologist Sergei Winogradsky and the Dutch microbiol- ogist and botanist Martinus Beijerinck, argued that the prime focus of microbiology 4 1 Introduction should be the role of microbes in the turnover of matter on the planet (Dworkin 2012). Toward that goal, Winogradsky developed the “Winogradsky Column” to study how a large variety of bacteria interacts in nature to metabolize organic and sulfur compounds. The two Schools, Koch/Pasteur as opposed to Winogradsky/Beijerinck, represented different viewpoints. The former concentrated on diseases and a single causative agent, while the latter focused on the environment and the interaction within mixtures of microorganisms. The Koch/Pasteur school won the conflict, mainly because of its power in combatting infectious diseases. The students of the Koch/Pasteur school received the academic appointments, became the teachers of microbiology, got most of the research funds, and became the editors of the journals. As we will discuss below, it was not until the end of the twentieth century that the environmental school began to find its rightful place in microbiology. During most of the twentieth century, microbiological research continued to focus on isolating microbes that were causative agents of disease and studying their transmission and mechanism of pathogenesis. Subsequently and not less important, these pure culture techniques were applied as model microbial systems to discover the underlining principles of biochemistry and genetics. To achieve that goal, most research in microbiology was performed with a limited number of pure bacterial cultures under defined laboratory conditions. At that time, insufficient tools were available to study the ecology, natural phylogeny, and evolution of microbes. In spite of these difficulties, Rene Dubos et al. (1966) studied germ-free and specific- pathogen-free mice for over a decade, determining the interactions between microbiota and factors such as nutrition, stress, maternal care, housing conditions, social interactions and sanitation, on immune function and health over the life course. An important background for subsequent microbiome research was pioneer studies of symbiosis between specific bacteria and their hosts (Douglas 1994). One of the best studied of these symbioses is that between the squid Euprymna scolopes and the bacterium Vibrio fischeri (Septer 2019). Ed Ruby, Margaret McFall-Ngai, and their colleagues have shown how the symbiosis is initiated, maintained, and the benefits that each partner receive from the interaction (Boettcher and Ruby 1990; McFall-Ngai and Ruby 1991, 1998; Ruby and McFall-Ngai 1992; Montgomery and McFall-Ngai 1998; Graf and Ruby 1998; McFall-Ngai 2002; Aschtgen et al. 2019). Another well-studied bacteria/animal model symbiosis involves the intracellular symbiont Buchnera aphidicola and its host, the pea aphid Acyrthosiphon pisum. Paul Buchner (1886–1978) published the first comprehensive molecular study of the interaction of a bacterial symbiont (Buchnera) and its host (the pea aphid) (Buchner 1965). He suggested that the function of the endosymbionts was the synthesis of missing nutrients for the host. This initial study on Buchnera was followed by studies on symbionts of many groups of insects, pursued by numerous investigators, including Paul and Linda Baumann, Nancy Moran, Jennifer Wernegreen, and Angela Douglas (Unterman et al. 1989; Munson et al. 1991; Baumann et al. 2000; Moran and Baumann 2000; Ochman and Moran 2001; Wernegreen 2002; Douglas and Raven 2003). 1.1 History 5

The best-studied bacteria/plant symbiosis is the / symbiosis, which has a long history (Angus and Hirsch 2010; Ramírez-Bahena et al. 2016; Wang et al. 2018). Beijerinck, one of the fathers of environmental microbiology, discovered bacteria (members of the genus Rhizobium) inside the root nodules of leguminous plants, which carried out fixation (Beijerinck 1901). In addition to having discovered a biochemical reaction vital to fertility, Beijerinck revealed this archetypical example of symbiosis between plants and bacteria. Because of its obvious importance to agriculture, many scientists have researched this symbiosis. Some of the discoveries that influenced modern microbiome research are host specificity (Broughton et al. 2000; Perret et al. 2000), signaling (Batut et al. 2011; Oldroyd 2013), exchange of nutrients (Zahran 1999; White et al. 2007), and interdependent metabolism (Dakora 1995; Barsch et al. 2006) between symbiont and plant. Taxonomy provides scientists with essential information, enabling them to under- stand the relationships between organisms. For , taxonomy allows the reliable identification of microbial strains from clinical or environmental samples. Bacterial taxonomy was initiated in the late nineteenth century, when phenotypic characteristics were incorporated into bacterial description, including motility, growth requirements, morphology, properties, colony size and color, and chemical reactions (Moore et al. 2010). The advent of techniques for PCR and sequencing DNA, coupled with bioinformatics for analyzing the data opened the door for studying and understanding the vast microbial richness associated with animal and plant microbiomes (Woese and Fox 1977; Pace et al. 1986; Amann et al. 1995; Wilson and Blitchington 1996; Hugenholtz et al. 1998). One of the first to use this technology on the human body was David Relman (Kroes et al. 1999), who showed that DNA methods yielded a more diverse view of the subgingival bacterial microbiota than did classical cultivation methods. Some of the other pioneers who used nonculture molecular techniques to examine microbiomes are as follows: The Flint group determined the phylogenetic relationships of butyrate-producing bacteria in the human gut (Barcenilla et al. 2000). The Gordon group analyzed the bacterial community of the intestine (Hooper et al. 2001). Rohwer et al. (2002) examined the bacterial diversity associated with a Caribbean coral, and he reintroduced the term holobiont to describe the coral host and all associated prokaryotic and eukaryotic microorganisms and viruses. Lindow and Brand (2003) described the microbiology of plant phyllospheres. The Blaser group reported on the bacteria in the human distal esophagus (Pei et al. 2004). I offer my apologies to the many other important early experimentalists in this field whom I do not mention here. From a theoretical point of view, Lynn Margulis (1970, 1990, and 1991) has often been credited as the pioneer in this field, with her concept of the holobiont and the origin of eukaryotic cells. However, Baedke et al. (2020) has recently argued that the German theoretical Adolf Meyer-Abich introduced the holobiont concept much earlier than Margulis (Meyer-Abich 1943). One of the reasons that Meyer- Abich’s contribution was not appreciated was that, with one exception (Meyer- Abich 1964), all of his publications were in German. Another reason was that at 6 1 Introduction the time there was no molecular evidence to support his claims. More recently, the hologenome concept of evolution provided a framework for microbiome discoveries (Rosenberg et al. 2007; Zilber-Rosenberg and Rosenberg 2008; Gilbert et al. 2012; Rosenberg and Zilber Rosenberg 2013, 2018; Bordenstein and Theis 2015; Brooks et al. 2016; Roughgarden et al. 2018;Suárez 2020). Whereas only a few microorganisms were used in the twentieth century as simple model systems to uncover the principles of biochemistry and genetics, we now understand that the role of microorganisms in biology is much more basic. Bacteria are not only model systems, but are an important part of biology as a whole, and specifically part of all animals and plants. Realizing this has led to a paradigm change in how we view the biological world. As the Nobel Prize laureate, Josh Lederberg (2000) prophetically wrote: But our most sophisticated leap would be to drop the manichaean view of microbes-“We good; they evil.”

1.2 Definitions and Concepts

One cannot explain words without making incursions into the sciences themselves, as is evident from dictionaries; and, conversely, one cannot present a science without at the same time defining its terms. — Gottfried Wilhelm Leibniz

Bacterial Species The definition of a species for bacteria is controversial (Chan et al. 2012). DNA–DNA hybridization (DDH) is an experimental method, which indirectly measures the overall similarity between two genome sequences (McCarthy and Bolton 1963). DDH has been the “gold standard” for bacterial species demarcation as it provides a clear and objective numerical threshold for a species boundary, for which 70% DDH is widely used (Tindall et al. 2010). However, due to the labor-intensive and error-prone nature of DDH experiments, there has been a continuous demand for an alternative genotype-based standard (Stackebrandt et al. 2002). Currently, the most widely used bacterial species defini- tion is a group of strains showing over 97% of 16S rRNA gene sequence identity (Wayne et al. 1987; Edgar 2018). Data analysis in such studies typically assigns 16S rRNA sequences to Operational Taxonomic Units (OTUs).

Fecal Microbiota Transplantation (FMT) Administration of the entire microbial community from healthy donor stool into the recipient’s intestinal tract to normalize or modify intestinal microbiota composition and function (Macconnachie et al. 2009; Aroniadis and Brandt 2013).

Holobiont Margulis (1991) and Mindell (1992) independently introduced the term holobiont to describe a host and its primary symbiont. The meaning was subse- quently expanded to include the host plus all of its symbiotic microorganisms, including viruses (Rohwer et al. 2002). A holobiont now refers to an animal or 1.2 Definitions and Concepts 7 plant host together with all associated microorganisms. Meta-organism (Bell 1998; Biagi et al. 2012) is a synonym for holobiont. I prefer holobiont because it is derived from the Greek word holos, which means whole or entire, whereas meta means higher or beyond, like metaphysics.

Hologenome The union of all the genes in the holobiont; all the genes in the microbiome plus the genes of the host constitute the hologenome (Rosenberg et al. 2007; Zilber-Rosenberg and Rosenberg 2008). The term metagenome is often used in the literature to describe the sum of the genetic information of an environmental sample, including an animal or plant host and its symbiotic microorganisms. How- ever, we consider hologenome to be a more appropriate term for describing the total genomes of an animal or plant for three reasons. First, metagenome is a general term for all the genetic material retrieved from any environmental sample, whereas hologenome is specific to the genetic material of a holobiont. Second, the prefix meta- (from Greek: μετά ¼ “after”, “beyond”, “adjacent”, “self”) is used in English to indicate a concept which is an abstraction from another concept, used to complete or add to the latter, such as in metaphysics, whereas the prefix holo- (from Greek: holos ¼ whole) is more appropriate because it is used in English to denote whole; entire; entirety. Third, the term hologenome parallels the generally accepted term holobiont.

Macrobes Animals and plants. Notation was introduced by O’Malley and Dupré (2007). Macrobe host refers to the monogenomic individual that derives from a zygote.

Metabolomics This term describes the analytical approaches used to determine the metabolite profile(s) in any given strain or single tissue. The resulting census of all metabolites present in any given strain or single tissue is called the metabolome (Griffin and Vidal-Puig, 2008).

Microbiome Following Lederberg and McCray (2001), a microbiome refers to all the symbiotic microorganisms, including Bacteria, , algae, fungi, and viruses, which share the “body space” of a host. Others have referred to microbiomes as the genomes of our affiliated microbial partners (Backhed et al. 2005; Turnbaugh et al. 2007). The term microbiota preceded the term microbiome and by some is considered synonymous to it. We refer to microbiota as being the microbes associated with an animal or plant, but not necessarily the entire community of microbes. The term microbiome was coined long before Lederberg and McCray popularized it (Prescott 2017). Whipps et al. (1988) wrote, “A convenient ecological framework in which to examine biocontrol systems is that of the microbiome. This may be defined as a characteristic microbial community occupying a reasonably well defined habitat, which has distinct physio-chemical properties. The term thus not only refers to the microorganisms involved but also encompasses their theatre of activity.” This 8 1 Introduction early, often forgotten, definition of microbiome is directly in line with its current usage in microbiology.

Microbiomology A new term defined as the study of microbiomes.

Microflora This term is often misused to describe microbiota. It originated at a time when bacteria were considered plants. Microflora are microscopic plants, not bacteria.

Mycobiome A combination of the words “” and “microbiome” used to refer to the fungal community of a microbiome (Ghannoum et al. 2010).

OTU An operational taxonomic unit (OTU) is an operational definition used to classify groups of closely related individuals (Sokal and Siseath, 1982; Nguyen et al. 2016).16S rRNA gene sequences can be clustered according to their similarity to one another, and OTUs are defined based on the similarity threshold (usually 97% similarity for bacterial species).

Phage Therapy Specific bacterial viruses (, or phages for short) are employed to kill specific bacterial pathogens.

Prebiotics Substrates that are selectively used by host microorganisms conferring a health benefit (Gibson et al. 2017).

Probiotics Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hoffman et al. 2008; Reid et al. 2019).

Species A group of interbreeding or potentially interbreeding organisms (Mayr 1942). Because this criterion cannot generally be applied to prokaryotes, the 97% identity of 16S rRNA genes is routinely used to define a species.

Superorganism The term has incorrectly been used in some scientific publications and the popular press to describe holobionts (Youle et al. 2013). However, as discussed by Youle et al. (2013), a superorganism is a colony of eusocial insects, such as ants (Holldobler and Wilson 2008). The “super” in superorganism denotes a higher level of organization, an association composed of multiple organisms of the same species, whereas holobionts are constructed from different species.

Symbiosis Anton de Bary (1879) first coined the term symbiosis as “the living together of different species.” This broad definition is still generally accepted. The symbiotic system is constructed from a large partner termed the host and smaller partners called symbionts. This arbitrary division by dimension between host and symbiont may not fit all systems because size can be measured by cell number or by genome size, and in the case of many symbioses the microorganisms both outnum- ber their host and contain more genetic information. In spite of these limitations, the References 9 definition of host and symbiont based on size continues to be widely used, although it would be more appropriate to consider both the host and the symbionts as constituents of the holobiont. Endo- and exo-symbionts refer to those living inside or outside host cells, respectively. Microbes that simply pass through the host are not considered symbionts. Pathogens, like Mycobacterium tuberculosis, which live with their host for decades (Stutz et al. 2018), are considered symbionts, whereas pathogens like Vibrio cholera, which kill their host or depart in a few days, would not be considered symbionts (Roughgarden et al. 2018). Symbiosis takes different forms: Commensalism is a relationship benefiting one party, while the other is unaffected; mutualism is a relationship benefiting both parties; and parasitism is a relationship benefiting one party to the other’s detriment.

Synbiotics Nutritional supplements combining prebiotics and probiotics in a syn- ergistic manner, hence synbiotics (Delphine et al. 2008; Mohanty et al. 2018).

Vertical Transmission The direct transfer of a microorganism from parent to offspring without mixing with microbes in the environment (Roughgarden et al. 2018).

Viable-But-Not-Culturable (VBNC) state A bacterium in the VBNC state refers to a cell, which can be demonstrated to be viable, while being incapable of undergoing the sustained cellular division required for growth in or on a medium normally supporting growth of that bacterium (Oliver 2010).

Virome The collection of bacterial and eukaryotic viruses found in the microbiome. The term describes the collection of nucleic acids, both RNA and DNA that make up the viral community associated with a particular ecosystem or organism (McDaniel et al. 2008; Garmaeva et al. 2019).

References

Amann, R. I., Ludwig, W., & Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59, 143–169. Angus, A. A., & Hirsch, A. M. (2010). Insights into the history of the legume-betaproteobacterial symbiosis. Molecular Ecology, 19,28–30. Aroniadis, O. C., & Brandt, L. J. (2013). Fecal microbiota transplantation: Past, present and future. Current Opinion in Gastroenterology, 29(1), 79–84. Aschtgen, M., Brennan, C. A., Nikolakakis, K., et al. (2019). Insights into flagellar function and mechanism from the squid–vibrio symbiosis. Microbiomes, 5, 32. https://doi.org/10.1038/ s41522-019-0106-5. Backhed, F., Ley, R. E., Sonnenburg, J. L., et al. (2005). Host-bacterial mutualism in the human intestine. Science, 307, 1915–1920. Baedke, J., Fábregas-Tejeda, A., & Nieves Delgado, A. (2020). The holobiont concept before Margulis. J Exp Zool (Mol Dev Evol), 334,1–7. https://doi.org/10.1002/jez.b.22931. 10 1 Introduction

Barcenilla, A., Pryde, S. E., Martin, J. C., et al. (2000). Phylogenetic relationships of butyrate- producing bacteria from the human gut. Appl Envion Microbiol, 66, 1654. https://doi.org/10. 1128/AEM.66.4.1654-1661. Barsch, A., Carvalho, H. G., Cullimore, J. V., et al. (2006). GC-MS based metabolite profiling implies three interdependent ways of ammonium assimilation in Medicago truncatula root nodules. Journal of Biotechnology, 127(1), 79–83. Batut, J., Mergaert, P., & Masson-Boivin, C. (2011). Peptide signaling in the rhizobium-legume symbiosis. Current Opinion in Microbiology, 14(2), 181–187. Baumann, P., Moran, N. A., & Baumann, L. (2000). Bacteriocyte-associated endosymbionts of insects. In M. Dworkin (Ed.), The prokaryotes (pp. 1–55). New York: Springer. Beijerinck, M. W. (1901). Über oligonitrophile Mikroben. Centralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abteilung II, 7, 561–582. Bell, G. (1998). Model metaorganism. A book review. Science, 282, 248. Biagi, E., Candela, M., Fairweather-Tait, S., et al. (2012). Ageing of the human metaorganism: The microbial counterpart. Age, 34, 247–267. Boettcher, K. J., & Ruby, E. G. (1990). Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. Journal of Bacteriology, 172(7), 3701–3706. Bordenstein, S. R., & Theis, K. R. (2015). Host biology in light of the microbiome: Ten principles of holobionts and hologenomes. PLoS Biology, 13, e1002226. pmid: 26284777. Brooks, A. W., Kohl, K. D., Brucker, R. M., et al. (2016). Phylosymbiosis: Relationships and functional effects of microbial communities across host evolutionary history. PLoS Biology, 14, e2000225. pmid: 27861590. Broughton, W. J., Jabbouri, S., & Perret, X. (2000). Keys to symbiotic harmony. Journal of Bacteriology, 182, 5641–5652. Buchner, P. (1965). Symbiosis in animals which suck plant juices. In Endosymbiosis of animals with plant microorganisms (pp. 210–432). New York: Interscience. Chan, J., Halachev, M. R., Loman, N. J., et al. (2012). Defining bacterial species in the genomic era: Insights from the genus Acinetobacter. BMC Microbiology, 12, 302. Dakora, F. D. (1995). A functional relationship between leghaemoglobin and nitrogenase based on novel measurements of the two proteins in legume root nodules. Annals of , 75(1), 49–54. de Bary, A. (1879). Die Erscheinung der Symbiose. Straßburg: Verlag Trubner. Delphine, M., Saulnier, A., Gibson, G. R., et al. (2008). In vitro effects of selected synbiotics on the human faecal microbiota composition. FEMS Microbiology Ecology, 66(3), 516–527. Dobell, C. (1932). Antony van Leeuwenhoek and his “little animals”. New York: Harcourt, Brace and Company. Douglas, A. E. (1994). Symbiotic Interactions (pp. 68–77). New York: Oxford Univ. Press. Douglas, A. E., & Raven, J. A. (2003). Genomes at the interface between bacteria and organelles. Phil Trans R Soc, Lond B, 358,5–18. Dubos, R., Savage, D., & Schaedler, R. (1966). Biological Freudianism. Lasting effects of early environmental influences. Pediatrics, 38(5), 789–800. Dworkin, M. (2012). Sergei Winogradsky: A founder of modern microbiology and the first microbial ecologist. FEMS Microbiology Reviews, 36, 364–379. Edgar, R. C. (2018). Updating the 97% identity threshold for 16S ribosomal RNA OTUs. Bioinfor- matics, 34(14), 2371–2375. Garmaeva, S., Sinha, T., Kurilshikov, A., et al. (2019). Studying the gut virome in the metagenomic era: Challenges and perspectives. BMC Biology, 17(1), 84. https://doi.org/10.1186/s12915-019- 0704-y. Ghannoum, M. A., Jurevic, R. J., Mukherjee, P. K., et al. (2010). Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathogens, 5, e1000713. https://doi.org/ 10.1371/journal.ppat.1000713. Gibson, G. R., Hutkins, R., Sanders, M. E., et al. (2017). Expert consensus document: The international scientific association for probiotics and prebiotics. (ISAPP) consensus statement on the definition and scope of prebiotics. Nature Reviews. Gastroenterology & Hepatology, 14, 491–502. References 11

Gilbert, S. F., Sapp, J., & Tauber, A. I. (2012). A symbiotic view of life: We have never been individuals. The Quarterly Review of Biology, 87, 325–341. Graf, J., & Ruby, E. G. (1998). Host-derived amino acids support the proliferation of symbiotic bacteria. Proc Natl Acad Sci (USA), 95(4), 1818–1822. Griffin, J. L., & Vidal-Puig, A. (2008). Current challenges in metabolomics for diabetes research: A vital functional genomic tool or just a ploy for gaining funding? Physiological Genomics, 34(1), 1–5. Hoffman, F. A., Heimbach, J. T., Sanders, M. E., et al. (2008). Executive summary: Scientific and regulatory challenges of development of probiotics as food and drugs. Clinical Infectious Diseases, 46, S53–S57. Holldobler, B., & Wilson, E. O. (2008). The superorganism: The beauty, elegance, and strangeness of insect societies. New York: W.W. Norton. Hooper, L. V., Wong, M. H., Thelin, A., et al. (2001). Molecular analysis of commensal host- microbial relationships in the intestine. Science, 291, 881–884. Hugenholtz, P., Goebel, B. H., & Pace, N. R. (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology, 180, 4765–4774. Koch, R. (1882). The etiology of tuberculosis. Berlin Clinical Weekly, 19, 221–230. Kroes, I., Lepp, P. W., & Relman, D. A. (1999). Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci (USA), 96, 14547–14552. Lederberg, J. (2000). Infectious history. Science, 288, 287–293. Lederberg, J., & McCray, A. T. (2001). ‘Ome sweet ‘omics—A genealogical treasury of words. Scientist, 15,8. Lindow, S. E., & Brand, M. T. (2003). Microbiology of the phyllosphere. Applied and Environ- mental Microbiology, 69, 1875–1883. Macconnachie, A. A., Fox, R., Kennedy, D. R., et al. (2009). Faecal transplant for recurrent Clostridium difficile-associated diarrhoea: A UK case series. The Quarterly Journal of Medi- cine, 102, 781–784. Margulis, L. (1970). Origin of eukaryotic cells. New Haven, CT: Yale University Press. Margulis, L. (1990). Words as battle cries: Symbiogenesis and the new field of endocytobiology. Bioscience, 40(9), 673–677. Margulis, L. (1991). Symbiosis as a source of evolutionary innovation: Speciation and morphogen- esis. In L. Margulis & R. Fester (Eds.), Symbiogenesis and Symbionticism (pp. 1–14). Cambridge: MIT Press. Mayr, E. (1942). Systematics and the origin of species. New York: Columbia Univ. Press. McCarthy, B. J., & Bolton, E. T. (1963). An approach to the measurement of genetic relatedness among organisms. Proc Natl Acad Sci (USA), 50, 156–164. McDaniel, L., Breitbart, M., Mobberley, J., et al. (2008). Metagenomic analysis of lysogeny in Tampa Bay: Implications for prophage gene expression. PLoS One, 3(9), e3263. https://doi.org/ 10.1371/journal.pone.0003263. PMC 2533394. McFall-Ngai, M. J. (2002). Unseen forces: The influence of bacteria on animal development. Developmental Biology, 242,1–14. McFall-Ngai, M. J., & Ruby, E. G. (1991). Symbiont recognition and subsequent morphogenesis as early events in an animal–bacterial symbiosis. Science, 254, 1491–1494. McFall-Ngai, M. J., & Ruby, E. G. (1998). Squids and vibrios: When they first meet. Bioscience, 48, 257–265. Meyer-Abich, A. (1943). Beiträge zur Theorie der evolution der Organismen. I. Das typologische Grundgesetz und seine Folgerungen für Phylogenie und Entwicklungsphysiologie (contributions to the evolutionary theory of organisms: I. the basic typological law and its implications for phylogeny and developmental physiology). Acta Biotheoretica, 7,1–80. Meyer-Abich, A. (1964). The historico-philosophical background of the modern evolution-biology. Bibliotheca Biotheoretica, 10. Leiden: E.J. Brill. Mindell, D. P. (1992). Phylogenetic consequences of symbioses: Eukarya and eubacteria are not monophyletic taxa. Biosystems, 27,53–62. 12 1 Introduction

Mohanty, D., Misra, S., & Mohapatra, S. (2018). Prebiotics and synbiotics: Recent concepts in nutrition. Food Bioscience, 26, 152–160. Montgomery, M. K., & McFall-Ngai, M. J. (1998). Late postembryonic development of the symbiotic light organ of Euprymna scolopes (Cephalopoda: Sepiolidae). Biological Bulletin, 195, 326–336. Moore, E. R., Mihaylova, S. A., Vandamme, P., et al. (2010). Microbial systematics and taxonomy: Relevance for a microbial commons. Research in Microbiology, 161, 430–438. Moran, N. A., & Baumann, P. (2000). Bacterial endosymbionts in animals. Current Opinion in Microbiology, 3, 270–275. Munson, M. A., Baumann, P., Clark, M. A., et al. (1991). Evidence for the establishment of aphid- eubacterium endosymbiosis in an ancestor of four aphid families. Journal of Bacteriology, 173, 6321–6324. Nguyen, N., Warnow, T., Pop, M., et al. (2016). A perspective on 16S rRNA operational taxonomic unit clustering using sequence similarity. npj Biofilms Microbiomes, 2, 16004. https://doi.org/ 10.1038/npjbiofilms.2016.4. O’Malley, M., & Dupré, J. (2007). Size doesn’t matter: Towards a more inclusive philosophy of biology. Biology and Philosophy, 22, 155–191. Ochman, H., & Moran, N. A. (2001). Genes lost and genes found: Evolution of bacterial pathogen- esis and symbiosis. Science, 292(5519), 1096–1099. Oldroyd, G. E. (2013). Speak, friend, and enter: Signaling systems that promote beneficial symbi- otic associations in plants. Nature Reviews Microbiology, 11, 252–264. Oliver, J. D. (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Reviews, 34, 415–425. Pace, N. R., Stahl, D. A., Lane, D. J., et al. (1986). The analysis of natural microbial populations by ribosomal RNA sequences. Advances in , 9,1–55. Pei, Z., Bini, E. J., Yang, L., et al. (2004). Bacterial biota in the human distal esophagus. Proc Natl Acad Sci (USA), 101, 4250–4255. Perret, X., Staehelin, C., & Broughton, W. J. (2000). Molecular basis of symbiotic promiscuity. Microbiology and Molecular Biology Reviews, 64, 180–201. Prescott, S. L. (2017). : Origin of the term microbiome and why it matters. Human Microb J, 4,24–25. Ramírez-Bahena, M. H., Peix, A., Velazquez, E., et al. (2016). History of research on legume- rhizobia symbiosis: A teaching approach. Res Gate, 19(1). Reid, G., Gadir, A. A., & Dhir, R. (2019). Probiotics: Reiterating what they are and what they are not. Frontiers in Microbiology, 10, 424. Rohwer, F., Seguritan, V., Azam, F., et al. (2002). Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series, 243,1–10. Rosenberg, E. (2017). It’s in your DNA. Oxford, UK: Elsevier Publishers. Rosenberg, E., & Zilber-Rosenberg, I. (2013). The Hologenome concept: Human, animal and plant microbiota. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319- 04241. Rosenberg, E., & Zilber-Rosenberg, I. (2018). The hologenome concept of evolution-after ten years. Microbiome, 6, 78. https://doi.org/10.1186/s40168-018-0457-9. Rosenberg, E., Koren, O., Reshef, L., et al. (2007). The role of microorganisms in coral health, disease and evolution. Nature Reviews. Microbiology, 5, 355–362. Roughgarden, J., Scott, F., Gilbert, S. F., Eugene Rosenberg, E., et al. (2018). Holobionts as units of selection and a model of their population dynamics and evolution. Biological Theory, 13(1), 44–65. Ruby, E. G., & McFall-Ngai, M. J. (1992). A squid that glows in the night: Development of an animal-bacterial mutualism. Journal of Bacteriology, 174, 4865–4870. Septer, A. N. (2019). The Vibrio-squid symbiosis as a model for studying interbacterial competi- tion. mSystems, 4(3), e00108-19. https://doi.org/10.1128/mSystems.00108-19. References 13

Sokal, R. R., & Siseath, P. H. A. (1982). Principles of numerical taxonomy. San Francisco, CA: W. H. Freeman. Stackebrandt, E., Frederiksen, W., Garrity, G. M., et al. (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. International Journal of Systematic and Evolutionary Microbiology, 52, 1043–1047. Stutz, M. D., Clark, M. P., & Doerflinger, M. (2018). Mycobacterium tuberculosis: Rewiring host cell signaling to promote infection. Journal of Leukocyte Biology, 103(2), 259–268. Suárez, J. (2020). The stability of traits conception of the hologenome: An evolutionary account of holobiont individuality. HPLS, 42, 11. https://doi.org/10.1007/s40656-020-00305-2. Tindall, B. J., Rosselló-Móra, R., Busse, H. J., et al. (2010). Notes on the characterization of strains for taxonomic purposes. International Journal of Systematic and Evolution- ary Microbiology, 60, 249–266. Turnbaugh, P. J., Ley, R. E., Hamady, M., et al. (2007). The human microbiome project. Nature, 449, 804–810. Unterman, B. M., Baumann, P., & McLean, D. L. (1989). Pea aphid symbiont relationships established by analysis of 16S rRNAs. Journal of Bacteriology, 171, 2970–2974. Wang, Q., Jinge Liu, J., & Zhu, H. (2018). Genetic and molecular mechanisms underlying symbiotic specificity in legume-Rhizobium interactions. Frontiers in Plant Science, 9, 313. https://doi.org/10.3389/fpls.2018.00313. Wayne, L. G., Brenner, D. J., Colwell, R. R., et al. (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. International Journal of Systematic and Evolutionary Microbiology, 37, 463–464. Wernegreen, J. J. (2002). Genome evolution in bacterial endosymbionts of insects. National Review, 3, 850–861. Whipps, J. M., Lewis, K., & Cooke, R. C. (1988). Mycoparasitism and plant disease control. In N. M. Burge (Ed.), Fungi in biological control systems (pp. 161–187). Manchester: Manchester University Press. White, J., Prell, J., James, E. K., et al. (2007). Nutrient sharing between symbionts. Plant Physiol- ogy, 144(2), 604–614. Wilson, K. H., & Blitchington, R. B. (1996). Human colonic biota studied by ribosomal DNA sequence analysis. Applied and Environmental Microbiology, 62, 2273–2278. Woese, C. R., & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc Natl Acad Sci (USA), 74, 5088–5090. Youle, M., Knowlton, N., Rohwer, F., et al. (2013). Superorganisms and holobionts. Microbe, 84, 152–153. Zahran, H. H. (1999). Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiology and Molecular Biology Reviews, 63(4), 968–989. Zilber-Rosenberg, I., & Rosenberg, E. (2008). Role of microorganisms in the evolution of animals and plants: The hologenome theory of evolution. FEMS Microbiology Reviews, 32, 723–735. Composition of Microbiomes 2

Together, they are referred to as our microbiome—and they play such a crucial role in our lives that scientists like [Martin J.] Blaser have begun to reconsider what it means to be human. — Michael Specter

Abstract

All animals and plants are holobionts, containing abundant and diverse symbiotic microorganisms. Bacteria are found inside host cells (endosymbionts) and on the surfaces of different tissues (exosymbionts). The number of microbial symbionts is estimated by total (microscopic) and/or viable counts. Viable counts are generally one to three orders of magnitude lower than the total counts because of the viable-but-not culturable state. The most abundant and diverse animal and plant bacterial symbionts are present in the digestive tract, and associated with roots, respectively. Animals contain ca. 109 bacteria per gram-wet weight; in humans, this corresponds to ca. 1014 bacteria, which is similar to the total number of human cells. Primarily culture-independent DNA-based technology, 16S rRNA gene sequencing, or metagenomics determine the diversity of bacteria. Because of the large diversity of bacteria, there are several hundred times more unique genes in the human microbiome than are present in the human genome. In healthy humans, diverse bacteria are associated with most organs, including the mouth, throat, nasal cavity, small intestine, large intestine, lungs, and vagina. Although there have been great advances in culture-independent DNA analyses of microbiomes over the last decade, many problems remain, such as limits of detection, lack of positive and negative controls, and reproducibility.

# Springer Nature Switzerland AG 2021 15 E. Rosenberg, Microbiomes, The Microbiomes of Humans, Animals, Plants, and the Environment 2, https://doi.org/10.1007/978-3-030-65317-0_2 16 2 Composition of Microbiomes

Keywords

Bacterial diversity · Bacterial abundance · Human microbiome · 16S rRNA gene- targeted fluorescent probes · Wolbachia

Understanding any ecological system, whether it is a lake or the human gut, requires knowledge of the number of organisms that are present, and the species and their relative abundance. In the case of microbiomes, this means determining the total number of microbes and species that are present and their relative abundance. This chapter focuses on the bacterial communities associated with macrobes. Chaps. 6 and 7 discuss eukaryotic microorganisms and viruses in microbiomes, respectively.

2.1 Abundance of Microbes in Holobionts

In general, animals contain ca. 109 bacteria per gram-wet weight, which interestingly is similar to rich soil. Although it has often been asserted that the number of bacteria in the human microbiome (ca. 1014) is 10 times higher than the number of cells in the human body, the ratio is actually quite variable and closer to one (Sender et al. 2016). Especially, high concentrations of symbiotic bacteria are found in certain marine sponges, 1010 per gram-wet weight (Hentschel et al. 2006). Regarding plants, bacteria are by far the most numerous colonists of plant leaves, being found in numbers up to 108 cells per gram, sufficiently numerous to contribute to the behavior of the individual plants on which they live (Lindow and Brand 2003; Morella et al. 2018). The rhizosphere (close root area) of plants contains ca. 109 bacteria per gram soil (Sylvia et al. 2005), the highest concentration being attached to the root epidermis. The number of microbial symbionts of various hosts has traditionally been estimated by total (microscopic) and/or viable counts. We now know that the viable counts of most environmental samples, such as water and soil, are generally one to three orders of magnitude lower than the total counts (Li et al. 2014a, b). The same is true for many determinations of associated with animals and plants. For example, human skin contains ca. 2 Â 1010 and 1 Â 1012 viable and total counts, respectively (Grice et al. 2009), and coral tissues contain 1 Â 106 and 2 Â 108 viable and total counts per cm2, respectively (Koren and Rosenberg 2006). Exceptions to this generality are human feces (van Houte and Gibbons 1966) and cow rumen (Grub and Dehority 1976). In these systems, using optimized media and anaerobic cultur- ing techniques, Eller et al. (1971) and Lagier et al. (2015, 2018) achieved similar total and viable counts, namely, about 1 Â 1011 bacterial cells per gram-wet weight of feces or rumen content. The large difference between total and viable counts in environmental samples is generally attributed to the viable-but-not-culturable (VBNC) state of many bacteria. A bacterium in the VBNC state has been defined “as a cell which can be demonstrated to be viable, while being incapable of undergoing the sustained 2.2 Diversity of Microbes in Holobionts 17 cellular division required for growth in or on a medium normally supporting growth of that cell” (Oliver 2010; Capozzia et al. 2016). In the cases of human gut and cow rumen bacteria, I suggest that the rapid turnover of bacteria in the gut and rumen would eliminate cells in the VBNC state, leaving only growing bacteria that can form colonies under appropriate conditions. Another possibility, not excluding the first, is the special effort put into growing bacteria in these two well-studied important systems. Abundance of specific groups of bacteria can be determined by combining microscopy with 16S rRNA gene-targeted fluorescent probes (Levsky and Singer 2003; Restrepo-Ortiz et al. 2018). For example, the number of Escherichia coli in a sample was determined by using an oligonucleotide probe that is complementary to that part of the 16S rRNA gene that is unique to E. coli (Smati et al. 2013). Choosing appropriate probes, enables determination of the abundance of specific strains, species, genera, phyla, or domains. This technology has been used widely, including with samples from human feces (Franks et al. 1998; Weickert et al. 2011; Alcon- Giner et al. 2017), chickens (Zhu and Joerser 2003), pigs (Hein et al. 2008), white flies (Gottlieb et al. 2008), honeybees (Romero et al. 2019), soybeans (Mitter et al. 2017), and rice roots (Lu et al. 2006).

2.2 Diversity of Microbes in Holobionts

Current research on the diversity of Bacteria and Archaea associated with a particu- lar organism relies primarily on culture-independent DNA-based technology (Hamady and Knight 2009; Pollock et al. 2018; Mailhe et al. 2018; Kai et al. 2019). The most popular approach relies on extracting the total DNA from a tissue, amplifying the 16S rRNA bacterial genes by polymerase chain reaction (PCR) technology and sequencing these genes. Sequences with greater than 97% identity are typically assigned to the same species, those with >95% identity are typically assigned to the same genus, and those with >80% identity are typically assigned to the same phylum (Kampfer and Glaeser 2013), although these definitions are unsupported and disputable (Schloss and Handelsman 2005; Beye et al. 2018). In sexually reproducing animals and plants, the ability of two organisms to interbreed and produce fertile offspring of both sexes is generally accepted as a simple indicator that the organisms share enough genes to be considered members of the same species. Thus, a “species” is a group of interbreeding or potentially interbreeding organisms (Mayr 1942). Because this criterion cannot generally be applied to prokaryotes, the 97% identity of 16S rRNA genes is routinely used to define a species. To be more precise, bacteria, which contain 16S RNA gene sequences with greater than 97% identity, are considered to be in the same operational taxonomic unit (OTU). However, it is interesting to consider that the conserved region of the 18S rRNA genes of man and chimpanzee show 99.2% identity (Laudien et al. 1985), so it is likely that different bacterial species may share more than 98% identity. As a part of the rebirth of culture techniques in microbiology, the use of multiple culture conditions combined with prolonged incubation time has permitted the 18 2 Composition of Microbiomes isolation of hundreds of new bacterial species from the human gut (Lagier et al. 2012, 2016, 2018). More recently, culturing was used to identify bacterial spores by first incubating fecal samples with ethanol to destroy vegetative bacteria and enrich the relative number of spores. This technique enabled the culture of 137 bacterial species, including 69 new bacterial taxa (Browne et al. 2016). The data indicated that ~55% of the bacterial genera in the of healthy individuals were able to produce resilient spores. Overall, these new culturing methods have more than doubled the number of bacterial species that have been isolated from the human gut. Isolation allows for the investigation of the phenotypes and functions of these isolates. I should also point out that Archaea, one of the three domains of life, are ubiquitously present in most, if not all, microbiomes associated with eukaryotic hosts. Archaea are interactive components of complex microbiomes (Moissl- Eichinger et al. 2019). However, they have been mostly overlooked in microbiome studies due to the lack of standardized detection protocols and to the fact that no archaeal pathogen is currently known (Bang and Schmitz 2018). The bacterial compositions of hundreds of different animal and plant species have been published during the last 20 years. Examples are presented in Tables 2.1, 2.2, and 2.3. The number of reported bacterial species associated with its host are minimum values because rare bacteria are not detected by current methods.

2.3 Microbiota of Invertebrates (Table 2.1)

Sponges, which are the oldest animal that still exists (Mehbub et al. 2014), range in size from a few millimeters to more than a meter in diameter. It is interesting that these filter-feeding sessile organisms, which may be the most ancient animal that still exists, contain the highest recorded concentration of microbial symbionts. Microbes can comprise as much as 40% of sponge tissue volume, with densities in excess of 109 microbial cells per ml of sponge tissue. It has been reported that sponges contain 5000 bacterial species, assigned to 25 bacterial phyla (Webster and Thomas 2016; Thomas et al. 2016). Sponge-associated microorganisms are generally present in the sponge extracellular matrix. However, some sponge species also host endosymbionts. Using 33 sponge species, belonging to 19 families and 5 orders, it was shown that both the diversity and the structure of sponge microbiomes are highly specific to host phylogeny at the order and family levels (Yang et al. 2019). Most research on the relationship between sponges and their associated microorganisms have studied only marine sponges. However, Laport et al. (2019) have recently reported that freshwater sponges also have a rich microbiota, compris- ing at least 44 phyla belonging mainly to Proteobacteria and low percentages of Bacteroidetes, Acidobacteria, and Verrucomicrobia. Sponge/bacteria symbioses benefit both partners. The hosts benefit from a wide range of metabolic processes, including nitrogen fixation (Feng and Li 2019), provided by the symbiont community, which helps remove waste products and provide nutrients. The symbionts benefit from a stable supply of nutrients and waste products, which are excreted by the host. Sponges also participate in marine