NMR spectra of Cephalotes ants gut and cuticle

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.

Methods
1H 1D and 2D NMR spectra were acquired on a Bruker Avance III HD spectrometer at 700.13 MHz, equipped with a 1H/13C/15N/2H 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 H2O was applied during the recycling time. Quantitative 1H 1D NMR spectra, were recorded with a recycling time d1 of 25 s (as 5 times the highest T1 1H), in quadriplicate with TSP-d4 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-1) 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.

15N 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. 1H-15N cross-polarization (CPMAS) experiments were performed at a MAS frequency of 14286 Hz with a contact time of 1 ms (1H and 15N 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 15N spectra were referenced using L-tyrosine (d = 40.4 ppm). The 1H-15N-13C double CP-MAS experiment was carried out on a Bruker Advance Neo spectrometer operating at 14.1 T (13C Larmor frequency of 150.9 MHz) using a 3.2 mm Bruker CPMAS CryoProbeTM 32. The MAS frequency was 11kHz, and the 1H-15N and 15N-13C contact times were 1.5 ms and 7 ms, respectively. The recycle delay was 3.5 s, and the 65536 scans were accumulated.