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bioRxiv preprint doi: https://doi.org/10.1101/526038; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Ecological specificity of the metagenome in a set of lower termite 2 species supports contribution of the microbiome to adaptation of the 3 host 4 5 Lena Waidele, Judith Korb, Christian R Voolstra, Franck Dedeine, and Fabian Staubach 6 7 8 Author affiliations 9 For L. Waidele, J. Korb, and F. Staubach see corresponding author address 10 11 Christian R Voolstra 12 Red Sea Research Center, Division of Biological and Environmental Science and 13 Engineering (BESE), King Abdullah University of Science and Technology (KAUST), 14 Thuwal 23955-6900, Saudi Arabia 15 [email protected] 16 17 Franck Dedeine 18 Institut de Recherche sur la Biologie de l’Insecte, UMR 7261, CNRS - Université François- 19 Rabelais de Tours, Tours, France 20 [email protected] 21 22 Corresponding author 23 Dr. Fabian Staubach 24 Biologie I 25 Universitaet Freiburg 26 Hauptstr. 1 27 79104 Freiburg 28 Germany 29 phone: +49-761-203-2911 30 fax: +49-761-203-2644 31 email: [email protected] 32 33 Running title 34 lower termite metagenomes 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1 bioRxiv preprint doi: https://doi.org/10.1101/526038; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 51 Originality-Significance Statement 52 This is the first study that applies metagenomic shotgun sequencing in a multispecies 53 framework of lower termites to gain insight into the contribution of eukaryote and bacterial 54 microbes to host adaptation. Our study design employs a selection of host species that 55 span different ecologies, reared under identical conditions, to tease apart the relative 56 contribution of acclimation vs. adaptive differences. Functional changes of the microbiome 57 that aligned with ecology were highly specific. We found the most prominent changes in 58 the bacterial metabolic metagenome, suggesting a role of the microbiome in dietary 59 adaptation. 60 61 Summary 62 Elucidating the interplay between hosts and their microbiomes in ecological adaptation has 63 become a central theme in evolutionary biology. The microbiome mediated adaptation of 64 lower termites to a wood-based diet. Lower termites have further adapted to different 65 ecologies. Substantial ecological differences are linked to different termite life types. 66 Termites of the wood-dwelling life type never leave their nests and feed on a uniform diet. 67 Termites of the foraging life type forage for food outside the nest, access a more diverse 68 diet, and grow faster. Here we reasoned that the microbiome that is directly involved in 69 food substrate breakdown and nutrient acquisition might contribute to adaptation to these 70 ecological differences. This should leave ecological imprints on the microbiome. To search 71 for such imprints, we applied metagenomic shotgun sequencing in a multispecies 72 framework covering both life types. The microbiome of foraging species was enriched with 73 genes for starch digestion, while the metagenome of wood-dwelling species was enriched 74 with genes for hemicellulose utilization. Furthermore, increased abundance of genes for 75 nitrogen sequestration, a limiting factor for termite growth, aligned with faster growth. 2 bioRxiv preprint doi: https://doi.org/10.1101/526038; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 These findings are consistent with the notion that a specific subset of functions encoded 77 by the microbiome contributes to host adaptation. 78 79 Introduction 80 The importance of microbes for the evolution of higher organisms is starting to be realized 81 (McFall-Ngai et al., 2013; Bang et al., 2018). Metazoan evolution is not only driven by 82 pathogenic microbes, as reflected by fast evolution of immune genes (Clark et al., 2007). 83 Rather, microbes often are facilitators of metabolic and environmental adaptations 84 (Jaenike et al., 2010; Douglas, 2015; Bang et al., 2018). 85 The gut microbial communities of wood-feeding roaches and termites facilitated 86 thriving on a wood diet that is difficult to digest and poor in nitrogen. Nitrogen fixation and 87 the digestion of wood depend on the termite gut microbiome (Brune, 2014; Brune and 88 Dietrich, 2015; Bang et al., 2018). In lower termites, lignocellulose degradation was initially 89 mainly attributed to unicellular eukaryotes (protists) in the gut (Cleveland, 1923). Recently, 90 it has become evident that lignocellulose degradation is a synergistic effort of the termite, 91 its associated protists, and bacteria (Scharf et al., 2011; Peterson et al., 2015; Peterson 92 and Scharf, 2016). Bacteria are also considered the major agents in reductive 93 acetogenesis (Leadbetter et al., 1999). In this process bacteria use hydrogen that is an 94 abundant by-product of cellulose degradation (Pester and Brune, 2007) as an energy 95 source, resulting in the production of acetic acid. Acetic acid, in return, serves the termite 96 host as an energy source (Brune, 2014). In addition to their role in acetic acid production, 97 bacteria are also essential for the fixation of atmospheric nitrogen (Lilburn et al., 2001; 98 Desai and Brune, 2012) and the recycling of nitrogen from urea or uric acid (Potrikus and 99 Breznak, 1981; Hongoh et al., 2008b). By using genome sequencing and pathway 100 reconstruction, these processes have been assigned to four major bacterial phyla in the 3 bioRxiv preprint doi: https://doi.org/10.1101/526038; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 termite gut: Proteobacteria (Desulfovibrio (Kuwahara et al., 2017)), Spirochetes 102 (Treponema (Warnecke et al., 2007; Ohkuma et al., 2015)), Bacteroidetes 103 (Azobacteroides) (Hongoh et al., 2008b) , and Elusimicrobia (Endomicrobium (Hongoh et 104 al., 2008a; Zheng et al., 2016)). 105 Many bacteria in the termite gut live in tight association with protists, where they sit 106 on the surface (Hongoh et al., 2007; Yuki et al., 2015), in invaginations of the cell 107 membrane (Kuwahara et al., 2017), or even inside the protist cells (Brune, 2012). Such 108 tight associations lead to frequent vertical transmission of bacteria between protist 109 generations. In return, protists and bacteria are vertically transmitted between termite 110 generations via proctodeal trophallaxis during colony foundation (Shimada et al., 2013). 111 Vertical transmission has led to co-speciation between bacteria and their protist hosts, and 112 sometimes even the termite hosts (Noda et al., 2007; Ohkuma, 2008; Ikeda-Ohtsubo and 113 Brune, 2009; Desai et al., 2010). Evidence for horizontal transfer of protists between 114 termite species, so called transfaunations, is limited to a few exceptions (Radek et al., 115 2018). Hence, the termite host species association is rather strict, leading to strong 116 phylogenetic imprints on protist community structure (Yamin, 1979; Kitade, 2004; Waidele 117 et al., 2017). In comparison, the bacterial microbiome is more flexible, frequently 118 transferred between termite host species (Bourguignon et al., 2018), and affected by diet 119 (Boucias et al., 2013; He et al., 2013; Dietrich et al., 2014; Mikaelyan et al., 2015; Rahman 120 et al., 2015; Rossmassler et al., 2015; Tai et al., 2015; Waidele et al., 2017). 121 There is evidence that the gut microbiome of termites has contributed to the 122 adaptation of different termite species to their specific ecologies (Zhang and Leadbetter, 123 2012; He et al., 2013; Waidele et al., 2017; Duarte et al. 2018, Liu et al., 2018). There are 124 pronounced ecological differences between the so-called termite life types (Abe, 1987; 125 Korb, 2007). Termite species of the wood-dwelling life type never leave their nest except 4 bioRxiv preprint doi: https://doi.org/10.1101/526038; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 126 for the mating flight. They feed on a uniform bonanza resource, that is the piece of wood 127 they built their nest in, and grow rather slowly (Lenz, 1994; Korb, Hoffmann, et al., 2012). 128 On the other hand, foraging species leave their nest to forage for food, can access a more 129 diverse diet, and grow faster (Lenz, 1994; Lainé et al., 2003). Different growth rates and 130 diets likely impose different selection pressures on the termite holobiont, in particular with 131 regard to nutrient uptake. Because the microbiome is directly involved in nutrient uptake, it 132 seems reasonable to hypothesize that it may also play a role in adaptation to life type 133 related ecological differences. In this scenario, one would expect the life types to leave an 134 imprint on microbiome structure and function. As such, searching for microbial imprints of 135 a given life type can possibly provide us with a lead for microbiome-mediated adaptation. 136 One potential pitfall of such an endeavor is that microbiomes may bear imprints 137 from transient microbes that were ingested from the environment. Transient microbes 138 rarely form evolutionary relevant relationships with the host (Douglas, 2011; Ma and 139 Leulier, 2018). Instead, they reflect the microbiome of the local environment the termites 140 were collected from. When the local environment correlates with other ecological 141 differences these imprints could be falsely interpreted as potentially adaptive changes of 142 the microbiome.