Comparative Genomic Analysis of the Endosymbionts of Herbivorous Insects Reveals Eco-Environmental Adaptations: Biotechnology Applications Weibing Shi 1,2 , Shangxian Xie 1,2,3 , Xueyan Chen 2,4 , Su Sun 1,2 , Xin Zhou 2 , Lantao Liu 1,2 , Peng Gao 1,2 , Nikos C. Kyrpides 5 , En-Gyu No 2 , Joshua S. Yuan 1,2 * 1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America, 2 Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, Texas, United States of America, 3 School of Life Sciences and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China, 4 Department of Veterinary Pathology, Texas A&M University, College Station, Texas, United States of America, 5 DOE Joint Genomes Institute, Walnut Creek, California, United States of America Abstract Metagenome analysis of the gut symbionts of three different insects was conducted as a means of comparing taxonomic and metabolic diversity of gut microbiomes to diet and life history of the insect hosts. A second goal was the discovery of novel biocatalysts for biorefinery applications. Grasshopper and cutworm gut symbionts were sequenced and compared with the previously identified metagenome of termite gut microbiota. These insect hosts represent three different insect orders and specialize on different food types. The comparative analysis revealed dramatic differences among the three insect species in the abundance and taxonomic composition of the symbiont populations present in the gut. The composition and abundance of symbionts was correlated with their previously identified capacity to degrade and utilize the different types of food consumed by their hosts. The metabolic reconstruction revealed that the gut metabolome of cutworms and grasshoppers was more enriched for genes involved in carbohydrate metabolism and transport than wood- feeding termite, whereas the termite gut metabolome was enriched for glycosyl hydrolase (GH) enzymes relevant to lignocellulosic biomass degradation. Moreover, termite gut metabolome was more enriched with nitrogen fixation genes than those of grasshopper and cutworm gut, presumably due to the termite’s adaptation to the high fiber and less nutritious food types. In order to evaluate and exploit the insect symbionts for biotechnology applications, we cloned and further characterized four biomass-degrading enzymes including one endoglucanase and one xylanase from both the grasshopper and cutworm gut symbionts. The results indicated that the grasshopper symbiont enzymes were generally more efficient in biomass degradation than the homologous enzymes from cutworm symbionts. Together, these results demonstrated a correlation between the composition and putative metabolic functionality of the gut microbiome and host diet, and suggested that this relationship could be exploited for the discovery of symbionts and biocatalysts useful for biorefinery applications. Citation: Shi W, Xie S, Chen X, Sun S, Zhou X, et al. (2013) Comparative Genomic Analysis of the Endosymbionts of Herbivorous Insects Reveals Eco- Environmental Adaptations: Biotechnology Applications. PLoS Genet 9(1): e1003131. doi:10.1371/journal.pgen.1003131 Editor: Paul M. Richardson, Progentech, United States of America Received April 10, 2012; Accepted October 15, 2012; Published January 10, 2013 Copyright: ß 2013 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research is funded by SouthCentral Sungrant and Texas Agrilife Bioenergy Research Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: syuan@tamu.edu Introduction Insects represent one of the most diverse groups of organisms on the planet that can adapt to the extremely diverse eco- environments. In particular, herbivorous insects can exploit a wide range of the plant species as food sources [1]. Insect gut symbionts play an essential role in the insect adaptation to various food types and they have been shown to be important for lignocellulosic biomass degradation, nutrient production, com- pound detoxification, and environmental adaptation [2–7]. Disrupting insect gut symbionts can significantly reduce the fitness of insects and can even cause serious diseases such as CCD (Colony Collapse Disease) [8]. Moreover, insect gut symbionts also were shown to be maternally inheritable from generation to generation, which suggests the symbiotic microbiota is a dynamic component of the competitive evolution between plants and herbivorous insects as well as a driving force for insect speciation [9,10]. For these reasons, insect gut symbionts have been the subject of extensive studies in recent years [10]. Previous studies highlighted several important features of some insect gut symbionts including their reduced genome size, convergent evolution, co-speciation, and complementary function with the host genome [11–15]. Recent studies also expanded our under- standing of the roles of insect gut symbionts in non-conventional functions like nitrogen recycling, reproductive manipulation, pigment production and many other aspects related to insect fitness [16,17]. Despite the progress toward understanding insect-symbiont relationships, there is still much to be learned especially with regard to facultative symbionts. Moreover, limited research has PLOS Genetics | www.plosgenetics.org 1 January 2013 | Volume 9 | Issue 1 | e1003131