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Genomic and Metagenomic Analyses of a Wood-Feeding Cockroach, Cryptocercus punctulatus
Department of Entomology
As readily available fossil fuels are depleted, attention has turned to lignocelluloses to convert solar energy into thermochemical energy. Lignocellulose is a naturally occurring complex of plant-derived materials that includes the hydrophilic sugar polymers cellulose and hemicellulose, and the hydrophobic organic glue lignin. However, to release the solar energy stored in plants through photosynthesis, an array of enzymes referred to as lignocellulases are required to disassociate a matrix of cellulose, hemicelluloses, pectins, lignin, and glycosidic linkages. Similar to lower termites, wood-feeding C. punctulatus and their gut symbionts have co-evolved into a suite of specialized enzymes that synergistically break down lignocelluloses.
While the pressing needs for bioenergy and biomass conversion have redirected termite research to focus more on the lignocellulose degradation, research on Cryptocercus has been primarily focused on evolutionary relationships with Isoptera (termites), and the development of sociality. There is virtually no information available on lignocellulose digestion and degradation in wood-feeding cockroaches. To fill this knowledge gap and to enrich the existing gene pool of lignocellulolytic enzymes, I propose to sequence 1) a cDNA library synthesized from the entire digestive tract without symbionts, and 2) a gDNA library extracted from hindgut microbial community of the wood-feeding cockroach using a high throughput 454 pyrosequencing platform.
The potential impacts of the proposed project are that
1) a complete genome-metagenome-wide survey of lignocellulases will add novel information regarding the enzymatic conversion of lignocellulosic biomass;
2) information gained from the wood-feeding cockroach may help to identify a subset of conserved lignocellulolytic enzymes throughout C. punctulatus "C R. flavipes lineage; the distribution of lignocellulase genes among different insects has yet to be explored in great detail. However, given the fact that Cryptocercus and lower termites share many flagellate symbionts such as oxymonadid and hypermastigid in their hindguts, it is germane to survey the homogeneity of lignocellulases across Dictyoptera with the basal C. punctulatus as a reference using a comparative genomic approach. Generally, evolutionarily conserved lignocellulases bear more biological meaning in the processes of lignocellulosic biomass conversion and may shed light on the core enzymes of the lignocellulose digestion machinery;
3) Cryptocercus maintains more diverse gut flagellate species than any existing termite species, and provides an invaluable resource for novel lignocellulases; and
4) it will help us to elucidate the operating mechanism underlying the host-symbiont lignocellulose digestion systems.
The role that endogenous and symbiotic lignocellulases play in lignocellulose digestion has remained a point of conjecture. The proposed sequencing will shed light on the genome organization and interactions (synergistically or independently) of the existing dual lignocellulase system in both C. punctulatus and R. flavipes.
2011 Project Description
The obligate relationship of both the wood-eating cockroach Cryptocercus and lower termites with lignocellulolytic flagellate protists in their hindguts has long been a textbook example of symbiosis. Cryptocercus is the sister group to Isoptera, and shares many similarities with lower termite taxa:
1) both feed on recalcitrant lignocelluloses (i.e., are xylophagous),
2) both exhibit an obligate relationship with hindgut symbionts, with many common flagellate genera co-occurring in both groups,
3) both are equipped with endogenous and symbiotic lignocellulases,
4) both transfer symbionts to conspecifics via proctodeal trophallaxis, and
5) both exhibit similar advanced social behaviors such as long-lasting biparental care.
Although research on Cryptocercus has always been intimately related to termites because of their intertwined relationships, the current research emphases of the two sister-taxa are different.
Wood-feeding insects have intrigued scientists for decades because of their unique capability of breaking down the matrix of cellulose, hemicelluloses, and lignin in plant cell walls. Lignocellulose depolymerization is a complex enzymatic process that involves an intricate collaboration between host organisms and their symbionts (pro- and eukaryotes). Although our knowledge of termite lignocellulose degradation is very limited, a recent metagenomic inventory of the microbiota from the hindgut of a higher termite sheds light on the potential role prokaryotic symbionts play in lignocellulose deploymerization, hydrogen metabolism, carbon dioxide reductive acetogenesis, and nitrogen fixation.
While the pressing needs for bioenergy and biomass conversion have redirected termite research to focus more on the lignocellulose digestion, research on Cryptocercus has been primarily focused on evolutionary relationships with Isoptera (termites), and the development of sociality. There is virtually no information available on lignocellulose digestion and degradation in woodroaches.
To fill this knowledge gap and to enrich the existing gene pool of lignocellulolytic enzymes, we sequenced and annotated 1) a cDNA library synthesized from the entire digestive tract including salivary gland, foregut, midgut, and hindgut (without symbionts), and 2) a genomic DNA (gDNA) library extracted from hindgut microbial community of a wood-eating cockroach, C. punctulatus, using a high-throughput 454 pyrosequencing platform.
Transcriptomic analysis of C. punctulatus digestive tract: Tissue-specific transcriptomes were sequenced at the University of Arizona Genetics Core. Salivary gland, foregut, midgut, and hindgut, respectively, from 30 adult roaches were dissected and pooled for the total RNA extraction. Each woodroach tissue accounted for 1/8 of a sequencing plate. The assemblies were accomplished using Roche 454's GS De Novo Assembler Software (version 2.3). A total number of unigenes (isotigs + singletons) from 4 tissue-based libraries are 82,805, 46,705, 102,784, and 112,821, respectively. BlastX database was used to annotate resulting isotigs. The cut-off E-value was 10-6. Sequences associated with carbohydrate catabolism were classified according to the CAZy nomenclature (http://www.cazy.org/).
Metatranscriptomic analysis of hindgut microbiota in C. punctulatus: Hindgut metatranscriptome were pyrosequenced by the Advanced Genetic Technologies Center (AGTC) at the University of Kentucky. Digestive tract from seven C. punctulatus adults (3 males, 4 females) was removed, and the luminal contents from hindgut were collected for sequencing. The total numbers of Reads and Bases from a half plate run are 1,010,243 and 228,975,825, respectively. The initial assembly step was to build a blast database of adapters used in the creation of library. After this, each read was blasted against this database using stringent settings to maximize the matching sequences. After blast was completed, the results were parsed and sorted in order to determine where each individual read should be trimmed. The results of this analysis were written in a format that allowed the creation of a new trimmed sff file from the original 454 run. This was done because Newbler does not take quality scores into consideration when using the FASTA files. After sequences trimmed and in sff format, Newbler was used to assemble the reads as normal. A total number of unigenes from symbiont library is 110,147. Based on the distribution profiles of carbohydrate active genes among different tissues and hindgut microbiota, endogenous lignocellulolytic enzymes are predominantly located at the salivary glands.
Sequence assembly and annotation: The main goal for 2011 was to annotate C. punctulatus digestive tissue-specific transcriptomes and hindgut metatranscriptome. The initial assembly and annotation have been concluded, and we now shift our focus to 1) carry out in-depth bioinformatic analysis of the entire woodroach digestome, 2) validate the initial annotation, manually curate, and clone the lignocellulolytic genes of interest, 3) functionally characterize selected lignocellulolytic enzymes in collaboration with Dr. Ling Yuan.
Liu, N., X. Yan, ML Zhang, L. Xie, Q. Wang, YP Huang, X. Zhou, SY Wang, ZH Zhou. 2011. Microbiome of fungus-growing termites: a new reservoir for mining lignocellulase genes. Appl. Environ. Micro. 77: 48-56.