GC×GC-TOFMS for the analysis of metabolites produced by termites (Reticulitermes flavipes) bred on different carbon sources

Abstract

More and more studies are dedicated to termites and their symbionts, to better understand how they efficiently produce energy from lignocellulose. In that context, a powerful analytical method was developed to perform the detection, separation and identification of compounds in the 1 µL fluid volume of the gut of the termite Reticulitermes flavipes. Comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS) was tested with three different column combinations: (1) low-polar/mid-polar; (2) polar/low-polar and (3) mid-polar/low-polar. The column set (3) offered the best separation and was chosen for further analysis and comparison study. Metabolites were detected in the samples, including amino acids, sugars, amines and organic acids. Samples collected from termites fed for 30 days on Avicel cellulose or xylan powder diets were analyzed and compared with the wood diet. Principal component analysis (PCA) of metabolite profiles demonstrated a separation of different clusters corresponding to the three different diets, with a similar trend for diets containing cellulose. The Analysis of variance (ANOVA) (one way-ANOVA and Tukey’s test) was used to compare compound levels between these three different diets. Significant differences were observed, including higher levels of aromatic derivatives in the wood diet and higher levels of sugar alcohols in the xylan diet. A higher accumulation of uric acid was observed with the artificial diets (cellulose and xylan), likely to be related to the nitrogen deficiency. The present study highlighted the capability of adaptation of the termite system to non-optimal carbon sources and the subsequent modification of the metabolite profile. These results demonstrate the potential interest to investigate metabolite profiling with state-of-the-art separation science tools, in order to extract information that could be integrated with other omics data to provide more insight into the termite-symbiont digestion system.