Biochemical and transcriptomic characterization of glycoside hydrolases in Thermobia domestica and Ctenolepisma longicaudata
Mallipeddi, R.  2018.  Ph.D. Dissertation.

The digestive system of phytophagous insects is considered a relevant prospecting resource for identification of novel cellulolytic enzymes that may improve industrial processes of cellulose degradation. While much is known from insect models of cellulose digestion, such as termites and roaches (Blattodea), there is a dearth of information on insects belonging to basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we detected species of Zygentoma as displaying the highest relative cellulolytic activity levels compared to all other tested insect orders, including Blattodea. The goals of the present study were to provide a morphohistological and biochemical characterization of the digestive system of Zygentoma, to annotate and screen for plant cell wall degrading enzyme (PCWDE) genes present in Thermobia domestica and Ctenolepisma longicaudata genomes, and to study their differential expression depending on diets with varying degree of cellulosic content. Morphohistological characterization supported no relevant differences in the digestive tube of T. domestica and C. longicaudata. Quantitative and qualitative cellulase assays identified the foregut as the region with the highest cellulolytic activity in both the tested insects, yet T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Annotation of predicted coding sequences from genomes of T. domestica and C. longicaudata reported numerous genes encoding for endoglucanases, glucosidases, β-1,3-glucanases, maltases, amylases, mannosidases, glucuronidases and lytic polysaccharide monoxygenases (LPMOs). Differential gene expression analysis of both species revealed that cellulase gene expression is primarily driven by type of tissue rather than diet. However, within each tissue of T. domestica and C. longicaudata, a higher number of PCWDEs and LPMOs were significantly up-regulated in paper, the most highly cellulosic diet, compared to all other tested diets. The annotation of coding sequences and differentially expressed PCWDE genes revealed highest identity to insect homologs, which suggests the potential conservation of PCWDEs through evolution and an ancient origin for cellulases in insects. These findings advance our understanding of cellulose digestion in a basal hexapod group and the identification of novel cellulolytic enzymes with potential application in the biofuel industry.