bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 Nitrogen conservation, conserved: 46 million years of N-recycling by the core symbionts of 2 turtle ants 3 4 Yi Hu1*, Jon G. Sanders2*, Piotr Łukasik1, Catherine L. D'Amelio 1, John S. Millar3, David R. 5 Vann4, Yemin Lan5, Justin A. Newton1, Mark Schotanus6, John T. Wertz6, Daniel J. C. 6 Kronauer7, Naomi E. Pierce2, Corrie S. Moreau8, Philipp Engel9, Jacob A. Russell1 7 8 1 Department of Biology, Drexel University, Philadelphia, PA 19104, USA 9 2 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 10 02138, USA 11 3 Department of Medicine, Institute of Diabetes, Obesity and Metabolism, University of 12 Pennsylvania, Philadelphia, PA 19104, USA 13 4 Department of Earth and Environmental Science, University of Pennsylvania, Philadelphia, PA 14 19104, USA 15 5 School of Biomedical Engineering, Science and Health systems, Drexel University, 16 Philadelphia, PA 19104, USA 17 6 Department of Biology, Calvin College, Grand Rapids, MI 49546, USA 18 7 Laboratory of Social Evolution and Behavior, The Rockefeller University, 1230 York Avenue, 19 New York, NY 10065, USA 20 8 Department of Science and Education, Field Museum of Natural History, Chicago, IL 60605, 21 USA 22 9 Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne, 23 Switzerland bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 24 * These authors contributed equally to this manuscript. 25 Category: Biological Sciences-Evolution 26 Key Words 27 Nutritional symbiosis, nitrogen metabolism, insects, metagenomics 28 Short title: A nitrogen-recycling symbiosis in turtle ants 29 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 30 Abstract 31 Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of 32 herbivory across the ants have raised expectations that nutritional symbionts have shaped their 33 diversification. Direct evidence for N-provisioning by internally housed symbionts is rare in 34 animals; among the ants, it has been documented for just one lineage. In this study we dissect 35 functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous 36 Cephalotes ants through in vivo experiments, (meta)genomics, and in vitro assays. Gut bacteria 37 recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are 38 acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes 39 species encode the pathways directing these activities, and several recycle N in vitro. These 40 findings point to a highly efficient N-economy, and a nutritional mutualism preserved for 41 millions of years through the derived behaviors and gut anatomy of Cephalotes ants. 42 43 Introduction 44 Nitrogen (N) is a key component of living cells and a major constituent of the nucleic acids and 45 proteins directing their structure and function. Like primary producers1, herbivorous animals face 46 the challenge of obtaining sufficient N in a world with limited accessible N, suffering 47 specifically due to the low N content of their preferred foods2. The prevalence of herbivory is, 48 hence, a testament to the many adaptations for sufficient N-acquisition. Occasionally featured 49 within these adaptive repertoires are internally housed, symbiotic microbes. Insects provide 50 several examples of such symbioses, with disparate herbivore taxa co-opting symbiont N- 51 metabolism for their own benefit3, 4, 5, 6. Such tactics are not employed by all insect herbivores7, 52 however, and few studies have quantified symbiont contributions to host N-budgets8, 9, 10, 11, 12. 53 bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. 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. 54 Ants comprise a diverse insect group with a broad suite of diets. Typically viewed as predators 55 or omnivores, several ants are functional herbivores, with isotopic N-ratios overlapping those of 56 known herbivorous insects13, 14. While occasionally obtaining N from tended sap-feeding insects, 57 most are considered plant canopy foragers, scavenging for foods such as extrafloral nectar, 58 pollen, fungi, vertebrate waste, and plant wound secretions14. Quantities of usable and essential 59 N in such foods are limiting15, 16. Hence, the repeated origins of functional herbivory provide a 60 useful natural experiment, enabling tests for symbiotic correlates of N-limited diets. The 61 concentration of specialized bacteria within herbivorous ant taxa suggests such a correlation17, 18. 62 But N-provisioning by internally housed symbionts has only been documented for carpenter ants, 63 whose intracellular Blochmannia provide them with amino acids made from recycled N19. 64 65 Herbivorous cephalotines (i.e. Cephalotes and Procryptocerus) and ants from other herbivore 66 genera (i.e. Tetraponera and Dolichoderus) exhibit hallmarks of a symbiotic syndrome distinct 67 from that in the Camponotini. Large, modified guts with prodigious quantities of extracellular 68 gut bacteria make up one defining feature20, 21, 22, 23, 24. Also characteristic are the oral-anal 69 trophallaxis events transmitting symbionts between siblings21, 25, 26, 27 and the domination of gut 70 communities by host-specific bacteria17, 28, 29. Such symbiotic “hotspots” stand out in relation to 71 several ant taxa, which show comparatively low investment in symbiosis18, 23. 72 73 N-provisioning by bacterial symbionts in these ants has been hypothesized as a mechanism for 74 their success in a seemingly marginal dietary niche14, 17. To investigate this, we focus on the 75 turtle ants of the genus Cephalotes (Fig. 1). With ~115 described species30, stable isotopes place 76 these arboreal ants low on the food chain14, 31. Workers are canopy foragers, consuming bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. 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. 77 extrafloral nectar and insect honeydew, fungi, pollen and leaf exudates32, 33, 34. Cephalotes also 78 consume mammalian urine and bird feces, excreta with large quantities of waste N accessible 79 only through the aid of microbes. Given this, the remarkably conserved gut microbiomes of 80 cephalotines28, 35 are proposed as an adaptation for their N-poor and N-inaccessible diets. Here 81 we measure symbiont N-provisioning in Cephalotes varians and gene content within the gut 82 microbiomes of 17 Cephalotes species (Table S1), describing symbiont N-metabolism across 46 83 million years of evolutionary history. 84 85 Results 86 Gut bacteria of turtle ants do not fix N2 36, 37, 38, 39, 40 87 Atmospheric N2-fixation is executed by bacterial symbionts of some invertebrates , 88 and prior detection of nitrogenase genes in ants17, 29 has led to the proposal that symbiotic 89 bacteria fix nitrogen for their hosts. To test this, three Cephalotes varians colonies were 90 subjected to acetylene reduction assays within hours of field capture. In three separate 91 experiments no ethylene was produced within test tubes containing acetylene-exposed ants 92 (Table S2), arguing against active N-fixation. 93 94 Symbiont-upgrading of dietary amino acids has minimal impact on workers’ N-budgets 95 Based on precedents from intracellular symbionts of insects12, 19, 41, we then tested whether gut 96 bacteria could upgrade dietary nitrogen compounds, transforming non-essential or inaccessible N 97 compounds into essential amino acids that are acquired by hosts. Our efforts focused on 98 glutamate, an important pre-cursor in the synthesis of many amino acids. Cephalotes varians 99 workers from three colonies were reared on artificial diets42 varying in the presence/absence of bioRxiv preprint doi: https://doi.org/10.1101/185314; this version posted September 7, 2017. 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. 100 antibiotics and the presence/absence of heavy isotope labeled glutamate. 13C or 15N were used to 101 label glutamate across our two separate experiments. Heavy isotope enrichment in the free amino 102 acid pools from worker hemolymph, assessed via GC-MS (Table S3), allowed us to quantify 103 symbiont glutamate upgrading and provisioning back to hosts. 104 105 Antibiotic treatment successfully suppressed gut microbial load in this and all below experiments 106 (Fig. S1), and workers survived treatments at rates sufficient for subsequent data generation (Fig. 107 S2). In addition, ants clearly absorbed nutrients from the administered diets, as hemolymph 108 glutamate pools showed 4-7% enrichment for heavy isotopes on the heavy vs. light isotope diets 109 in the absence of antibiotics (p=0.0033 15N vs. 14N diet; p=0.0018 13C vs. 12C diet). Yet, ant 110 acquisition of symbiont-processed C and N from dietary glutamate was minimal. For instance, 111 on the 13C-glutamate diet, antibiotic treatment reduced the fraction of heavy isotope-bearing 112 isoleucine (p=0.0147), leucine (p=0.0004), threonine (p=0.0029), and tyrosine (p=0.0169) in 113 worker hemolymph (Fig. S3), relative to estimates on this same diet without antibiotics. But 114 effect sizes for each amino acid were small, with changes of just 1.3-2.6% in ants with 115 suppressed microbiota.