Expression of Plant Cell Wall Degrading Enzymes and Digestive Physiology in Phasmatodea

Plant cell walls (PCWs) are the most abundant biomass on planet Earth. Unlike vertebrate animals, many invertebrates, especially insects, can digest PCWs, either to access the protoplasm or to break the wall itself into digestible, nutritive oligomers and monomers (Pauchet, Wilkinson et al. 2010, Calderon-Cortes, Quesada et al. 2012). This ability of many insects to break down recalcitrant PCWs has contributed greatly to their biodiversity and success around the world (Tokuda 2019). Traditionally, the breakdown of lignocellulosic and cellulosic material by insects was thought to be dependent on gastrointestinal microbes. Recent research with molecular and omics techniques (genomics, transcriptomics, and proteomics) has provided evidence to reveal PCW-degrading mechanisms in insects that use various arsenals of endogenous digestive enzymes. A few studies have reported on the endogenous PCW-degrading enzymes (PCWDEs) of Phasmatodea or stick insects. Much is still unknown with respect to how and where PCWDEs perform in a phasmid’s digestive tract, in addition to the question of whether gut microbial PCWDEs are present to contribute. This dissertation applies RNA-Seq and metagenomic analysis to identify the repertoire of endogenous PCWDEs in Aretaon asperrimus and Medauroidea extradentata, to assess the expression profiles of these enzymes through the alimentary canal, and to investigate the potential digestive role that could be played by gut microbes. We identify abundant endogenous PCWDE repertoires in each species, including GH9 endoglucanases, GH1 cellobiases, and GH28 polygalacturonases. Most PCWDE families are highly expressed in the anterior section of the midgut, while beta-glucosidases show a global expression pattern in multiple tissues, including the head. We observed very low expressions of GH9 and GH28 in hindgut tissues (ileum and rectum). Hemicellulases from GH2, GH3, and GH30 also showed global expression profiles without high expression in the AMG. The compartmentalization of phasmid digestion is investigated by an annotation and enrichment test for differentially expressed genes and the 1,000 most highly expressed genes in each tissue. The metagenomic study indicates that the dominant bacterial phylum, Proteobacteria, in both species’ gut communities, shows a great number of polygalacturonase sequences in the posterior section, which infers a potential pectin degradation synergism between endogenous and bacterial pectinases in different sections of a phasmid’s midgut. A lesser capability for PCW-degrading was observed in both species’ midgut microbiota, with a lack of cellulolytic Fibrobacteres and Spirochaetes. Overall, the identified transcripts of various glycoside hydrolase families in this dissertation further support that phasmids have uncommonly rich endogenous repertoires of PCWDEs. All these sequences could benefit future evolutionary analyses of these gene families and, more broadly, the phylogenetic analyses in Phasmatodea. Our results from the expression profiles of identified PCWDEs take a step further to illustrate how these endogenous digestive enzymes perform differently among tissues. The discovery of many bacterial PCWDEs in phasmid’s midgut in this dissertation indicates a possible synergism in the degradation of PCWs, which contradicts previous claims. All these findings contribute to the understanding of the digestive physiology in Phasmatodea and provide genomic data for future studies