DOI: 10.5061/dryad.d7wm37q0h
title: NMR spectra of Cephalotes ants gut and cuticle

description: Across the evolutionary history of insects, the shift from nitrogen-rich
 carnivore/omnivore diets to nitrogen-poor herbivorous diets was made
 possible through symbiosis with microbes. The herbivorous turtle ants
 Cephalotes possess a conserved gut microbiome which enriches the nutrient
 composition by recycling nitrogen-rich metabolic waste to increase the
 production of amino acids. This enrichment is assumed to benefit the host,
 but we do not know to what extent. To gain insights into nitrogen
 assimilation in the ant cuticle we use gut bacterial manipulation, 15N
 isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear
 magnetic resonance spectroscopy to demonstrate that gut bacteria
 contribute to the formation of proteins, catecholamine cross-linkers, and
 chitin in the cuticle. This study identifies the cuticular components
 which are nitrogen-enriched by gut bacteria highlighting the role of
 symbionts in insect evolution and provides a framework for understanding
 the nitrogen flow from nutrients through bacteria into the insect cuticle.
description: <sup>1</sup>H 1D and 2D NMR spectra were acquired on
 a Bruker Avance III HD spectrometer at 700.13 MHz, equipped with a
 <sup>1</sup>H<sup>/13</sup>C/<sup>15</sup>N/<sup>2</sup>H cryoprobe at a low temperature: 277 K to prevent any degradation of the metabolites. For all 1D and 2D experiments, a continuous wave presaturation on the residual H<sub>2</sub>O was applied during the recycling time. Quantitative <sup>1</sup>H 1D NMR spectra, were recorded with a recycling time d1 of 25 s (as 5 times the highest T1 <sup>1</sup>H), in quadriplicate with TSP-d<sub>4</sub> as an internal reference. For the 2D DOSY experiment a stimulated echo with bipolar gradients including a longitudinal eddy current delay was chosen. A 6k x 56 matrix was acquired, zero-filled to 16k x 56 and apodized in F2 by a decreasing exponential with 2 HZ line broadening. A recycling time d1 = 5 s was used as well as a diffusion time d20 = 200 ms, with a gradient length of 900 ms and 200 ms of resting delay, a linear gradient ramp was used from 2% to 95% of the maximum gradient amplitude (50 G.cm<sup>-1</sup>) and a number of accumulations NS = 200. An inverse Laplace transform was applied to obtain the 2D DOSY map displaying the diffusion coefficients along the indirect dimension. 2D COSY and ZQF-TOCSY spectra were also recorded. <sup>15</sup>N solid state NMR experiments were carried out on a Bruker Avance III spectrometer operating at 9.4 T (Larmor frequency of 40.5 MHz) using a 4 mm magic-angle spinning (MAS) double resonance probe head. Around 10 mg of cuticle per treatment (5 individuals) was packed in a 50µL HRMAS rotor to ensure a proper centering of the sample. <sup>1</sup>H-<sup>15</sup>N cross-polarization (CPMAS) experiments were performed at a MAS frequency of 14286 Hz with a contact time of 1 ms (<sup>1</sup>H and <sup>15</sup>N radiofrequency fields during CP of ca. 55 kHz and 40 kHz respectively), and with a recycling delay of 1 s. The experimental time was about 2-2.5 days for each spectrum. The <sup>15</sup>N spectra were referenced using L-tyrosine (d = 40.4 ppm). The <sup>1</sup>H-<sup>15</sup>N-<sup>13</sup>C double CP-MAS experiment was carried out on a Bruker Advance Neo spectrometer operating at 14.1 T (<sup>13</sup>C Larmor frequency of 150.9 MHz) using a 3.2 mm Bruker CPMAS CryoProbe<sup>TM</sup> <sup>32</sup>. The MAS frequency was 11kHz, and the <sup>1</sup>H-<sup>15</sup>N and <sup>15</sup>N-<sup>13</sup>C contact times were 1.5 ms and 7 ms, respectively. The recycle delay was 3.5 s, and the 65536 scans were accumulated.
description: The data sets are organized in main directories that correspond
 to the figures of Duplais et al, 2020. Directories '- Untreated ant
 guts -' and '- Treated ant guts -' contains liquid state
 NMR experiments for SI Fig 4-4 and SI Table S3. Each directory Fig. 1, Fig
 S8-10 S13-14 contains the solid-state NMR experiments. Within each of
 these directories, there is one directory for each spectrum acquired.
 These directories have extension .fid and their name specifies the
 experimental conditions. Each of these directories includes files that
 contain the raw NMR data (fid), the acquisition parameters (procpar), a
 log file (log), a text file with a more detailed description of
 experimental conditions (text). Below is a summary of the main
 directories. To view the files, please download the
 file Duplais_NMR_data.zip and uncompress it. Processed
 files with extension .fid or .ser are ready to be viewed in NMRViewJ or
 NMRDraw, respectively. In case of any questions, please
 do not hesitate to contact the authors. NMR
 datasets: <strong>Untreated ant
 guts</strong><br> Figure S5 and S6<br> 13: 2D ZQF-TOCSY
  (998 and 999: extracted rows)<br> Figure S3 and Table S3<br>
 17: Quantitative 1D NMR replicate #1<br> 18: Quantitative 1D NMR
 replicate #2<br> 19: Quantitative 1D NMR replicate #3<br> 20:
 Quantitative 1D NMR replicate #4<br> 21: Quantitative 1D NMR
 replicate #5<br> Figure S6<br> 24: 2D DOSY (98: processed 2D
 map, 9998 and 9999: extracted rows)
 <strong>Treated ant guts</strong><br> Figure
 S4<br> 20: 2D COSY<br> Table S3<br> 40: Quantitative 1D
 NMR replicate #1<br> 41: Quantitative 1D NMR replicate #2<br>
 42: Quantitative 1D NMR replicate #3<br> 43: Quantitative 1D NMR
 replicate #4<br> 44: Quantitative 1D NMR replicate #5
 Fig. 1 Cephalotes Non treated Labelled CP-MAS 15N NMR
 Fig. 1 Cephalotes Non Treated Unlabelled CP-MAS 15N NMR
 Fig. 1 Cephalotes Treated Labelled CP-MAS 15N NMR
 Fig. S8 Chitine-alpha CP-MAS 15N NMR Fig. S9
 Chitosan CP-MAS 15N NMR Fig. S10 Double CP-MAS 13C 15N
 NMR Fig. S13 Melanin_sepia CP-MAS 15N NMR
 Fig. S14 Melanin-synth CP-MAS 15N NMR