Treponema endosymbionts are the dominant bacterial members with ureolytic potential in the gut of the wood-feeding termite, Reticulitermes hesperus

49 Termites are remarkable for their ability to digest wood as their main energy source, but 50 the extremely low nitrogen (N) content of their diet presents a major challenge for N acquisition. 51 Besides the N 2 -fixing bacteria in the gut, the symbiotic groups that recycle N from waste 52 products as a complementary N-provisioning mechanism in termites remains poorly understood. 53 In this study, we used a combination of high-throughput amplicon sequencing, quantitative PCR, 54 and cultivation to characterize the microbial community capable of degrading urea, a common 55 waste product, into ammonia in the guts of two colonies of Reticulitermes hesperus termites. 56 The abundance of the ureC gene, which encodes for the alpha subunit of the urease enzyme, 57 ranged from 0.43 to 2.93% of the total prokaryotic community. Taxonomic analysis indicated 58 that 27.6% of the ureC gene amplicons in the termite gut matched with a Treponema 59 endosymbiont of gut protists previously found in several other termites, suggesting an important 60 contribution to the nutrition of essential cellulolytic protists. This corroborated our cultivation 61 efforts, where a majority of the isolates recovered had ureolytic potential and matched the other 62 taxa from our ureC gene sequences. Together, our results underscore a more important role for 63 ureolysis by endosymbionts within protists than by free-swimming bacteria in the gut lumen of 64 R. hesperus .


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Termites are social insects descended from wood-feeding cockroaches [1] and have long 74 been studied for their ability to digest lignocellulose [2,3]. This ability is driven by essential 75 contributions from deeply evolved, mutualistic symbionts found in their hindguts consisting of 76 archaea and bacteria in the 'higher termites' (Family Termitidae) and a tripartite community of 77 free-swimming archaea, bacteria, and cellulolytic protists with their ecto-and endosymbionts in 78 the evolutionarily basal 'lower termites' [2,3]. While biological nitrogen N2 fixation (BNF) by 79 bacterial symbionts is a prominent route of nitrogen (N) acquisition in termites to compensate 80 for their N-limited diet [1][2][3], BNF rates have been reported to be highly variable, suggesting that 81 some species with low BNF rates such as the Reticulitermes must rely on different pathways to 82 satisfy all host nutrient requirements [4]. Termites release most nitrogenous waste as uric acid, 83 and a previous study showed that gut bacteria in wood-feeding Reticulitermes flavipes termites 84 are capable of recycling N in uric acid for re-absorption into host tissue [5]. This process was 85 confirmed in several bacterial strains isolated from termites that can ferment uric acid to produce 86 ammonia [6,7]. However, uric acid can be converted into urea by enzymes produced by the host 87 or symbionts [8]; urea can be excreted as a waste product by protist cells [9]. At this step, 88 whether there are symbionts that can produce urease enzymes to catalyze the breakdown of 89 urea to ammonia for re-assimilation in the termite gut remains unknown. This represents a 90 knowledge gap in symbiont-mediated processes that may affect termite nutrition. To fill this gap, 91 we characterized the taxonomic diversity and abundance of symbionts with ureolytic potential 92 in the hindguts of two colonies of termites.

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Urec_98 shared a 96.61% similarity in the protein coding sequence with the ureC gene from 111 these endosymbionts and was genetically divergent from the sequences of the free-living 112 Treponema bryantii and T. ruminis (71.97% identity for both), indicating that Urec_98 is 113 evolutionarily distant from other Treponema species with ureolytic potential and likely an 114 endosymbiont of protists in the termite gut. The abundance of ureC gene ranged from 131 to 115 2,171 copies ng DNA -1 , which was 0.43 to 2.93% of the total prokaryotic community when 116 calculated as a proportion of the 16S rRNA gene copies ( Table 1). Out of a total of 192 isolates 117 retrieved from our cultivation procedures, most of the identified strains were from the phyla 118 Proteobacteria (148 isolates), Bacteroidetes (11 isolates) and Firmicutes (6 isolates) (Table S1).

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Approximately 92.2% of these isolates possessed ureolytic potential based on diagnostic PCR 120 of the ureC gene, and the most abundant identified strains were from the genus Citrobacter (88 5 and Bacillus (5 isolates) (Table S1). Taken together, our results demonstrate that the ureC gene 123 is present in the termite gut with a majority of the sequences represented by endosymbionts.

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Urec_98 was the single most abundant phylotype from our ureC gene dataset and is 125 closely related to "Candidatus Treponema intracellularis," an endosymbiont of Eucomonympha 126 protists previously found in wood-feeding Hodotermopsis sjoestedti termites [11]. The 127 "Candidatus T. intracellularis" genome contains genes encoding for urease as well as a               Phylogenetic characterization of Urec_98 based on protein-coding sequences of the ureC gene. Sequences from other Treponema species, bacteria previously isolated from termite guts, and other strains spanning major bacterial phyla for which data are available were included as reference species. The tree was constructed by the maximum-likelihood method using the Jones-Thorton-Taylor model.
Numbers at branch nodes indicate bootstrap support (500 replicates) above 50%, sequence accession numbers are given in parentheses, and the bar shows 0.1 substitutions per position.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TermiteUreaseManuscriptSupplementREVISEDFINAL.pdf