Transcriptome and microbiome of coconut rhinoceros beetle (Oryctes rhinoceros) larvae (original) (raw)

Uncovering the cultivable microbial diversity of costa rican beetles and its ability to break down plant cell wall components

PloS one, 2014

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, a, b and c-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.

Metagenomic Profiling Reveals Lignocellulose Degrading System in a Microbial Community Associated with a Wood-Feeding Beetle

PLoS ONE, 2013

The Asian longhorned beetle (Anoplophora glabripennis) is an invasive, wood-boring pest that thrives in the heartwood of deciduous tree species. A large impediment faced by A. glabripennis as it feeds on woody tissue is lignin, a highly recalcitrant biopolymer that reduces access to sugars and other nutrients locked in cellulose and hemicellulose. We previously demonstrated that lignin, cellulose, and hemicellulose are actively deconstructed in the beetle gut and that the gut harbors an assemblage of microbes hypothesized to make significant contributions to these processes. While lignin degrading mechanisms have been well characterized in pure cultures of white rot basidiomycetes, little is known about such processes in microbial communities associated with wood-feeding insects. The goals of this study were to develop a taxonomic and functional profile of a gut community derived from an invasive population of larval A. glabripennis collected from infested host trees and to identify genes that could be relevant for the digestion of woody tissue and nutrient acquisition. To accomplish this goal, we taxonomically and functionally characterized the A. glabripennis midgut microbiota through amplicon and shotgun metagenome sequencing and conducted a large-scale comparison with the metagenomes from a variety of other herbivoreassociated communities. This analysis distinguished the A. glabripennis larval gut metagenome from the gut communities of other herbivores, including previously sequenced termite hindgut metagenomes. Genes encoding enzymes were identified in the A. glabripennis gut metagenome that could have key roles in woody tissue digestion including candidate lignin degrading genes (laccases, dye-decolorizing peroxidases, novel peroxidases and βetherases), 36 families of glycoside hydrolases (such as cellulases and xylanases), and genes that could facilitate nutrient recovery, essential nutrient synthesis, and detoxification. This community could serve as a reservoir of novel enzymes to enhance industrial cellulosic biofuels production or targets for novel control methods for this invasive and highly destructive insect.

Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading cerambycid beetle

BMC Genomics, 2013

Background: Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of larval Anoplophora glabripennis, a wood-boring beetle with documented lignin-, cellulose-, and hemicellulose-degrading capabilities, which provides valuable insights into how this insect overcomes challenges associated with feeding in woody tissue. Results: Transcripts from putative protein coding regions of over 9,000 insect-derived genes were identified in the A. glabripennis midgut transcriptome using a combination of 454 shotgun and Illumina paired-end reads. The most highly-expressed genes predicted to encode digestive-related enzymes were trypsins, carboxylesterases, βglucosidases, and cytochrome P450s. Furthermore, 180 unigenes predicted to encode glycoside hydrolases (GHs) were identified and included several GH 5, 45, and 48 cellulases, GH 1 xylanases, and GH 1 β-glucosidases. In addition, transcripts predicted to encode enzymes involved in detoxification were detected, including a substantial number of unigenes classified as cytochrome P450s (CYP6B) and carboxylesterases, which are hypothesized to play pivotal roles in detoxifying host tree defensive chemicals and could make important contributions to A. glabripennis' expansive host range. While a large diversity of insect-derived transcripts predicted to encode digestive and detoxification enzymes were detected, few transcripts predicted to encode enzymes required for lignin degradation or synthesis of essential nutrients were identified, suggesting that collaboration with microbial enzymes may be required for survival in woody tissue.

An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

PLoS Genetics, 2010

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and wholegenome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydratedegrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.

Molecular approaches to study the insect gut symbiotic microbiota at the ‘omics’ age

Insect Science, 2010

Insect gut symbiotic microbiota play essential roles in the growth, development, pathogenesis and environmental adaptation of host insects. The molecular and systems level analysis of insect gut symbiotic microbial community will allow us to discover novel biocatalysts for biomass deconstruction and to develop innovative strategies for pest management. We hereby review the various molecular biology techniques as applied to insect gut symbiont analysis. This review aims to serve as an informative resource for experimental design and research strategy development in the field. We first discuss various strategies for sample preparation and their pros and cons. The traditional molecular techniques like DGGE, RFLP and FISH are covered with respect to how they are applied to study the composition, diversity and dynamics of insect gut symbiotic microbiota. We then focus on the various 'omics' techniques. The metagenome analysis together with the recent advancements in next-generation sequencing will provide enormous sequencing information, allowing in-depth microbial diversity analysis and modeling of pathways for biological processes such as biomass degradation. The metagenome sequencing will also enable the study of system dynamics and gene expression with metatranscriptome and metaproteome methods. The integration of different 'omics' level data will allow us to understand how insect gut works as a system to carry out its functions. The molecular and systems-level understanding will also guide the reverse design of next-generation biorefinery.

Characterization of the gut microbiota of invasive Agrilus mali Matsumara (Coleoptera: Buprestidae) using high-throughput sequencing: uncovering plant cell-wall degrading bacteria

Scientific Reports

the genus Agrilus comprises diverse exotic and agriculturally important wood-boring insects that have evolved efficient digestive systems. Agrilus mali Matsumara, an invasive insect, is causing extensive mortality to endangered wild apple trees in Tianshan. In this study, we present an in-depth characterization of the gut microbiota of A. mali based on high-throughput sequencing of the 16S rRNA gene and report the presence of lignocellulose-degrading bacteria. Thirty-nine operational taxonomic units (OTUs) were characterized from the larval gut. OTUs represented 6 phyla, 10 classes, 16 orders, 20 families, and 20 genera. The majority of bacterial OTUs belonged to the order Enterobacteriales which was the most abundant taxa in the larval gut. Cultivable bacteria revealed 9 OTUs that all belonged to Gammaproteobacteria. Subsequently, we examined the breakdown of plant cell-wall compounds by bacterial isolates. Among the isolates, the highest efficiency was observed in Pantoea sp., which was able to synthesize four out of the six enzymes (cellulase, cellobiase, β-xylanase, and β-gluconase) responsible for plant-cell wall degradation. One isolate identified as Pseudomonas orientalis exhibited lignin peroxidase activity. Our study provides the first characterization of the gut microbial diversity of A. mali larvae and shows that some cultivable bacteria play a significant role in the digestive tracts of larvae by providing nutritional needs.

Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

BMC Genomics, 2012

Background: The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results: Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin-and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (β-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level. Conclusions: Combining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.

Studying the organization of genes encoding plant cell wall degrading enzymes in C hrysomela tremula provides insights into a leaf beetle genome

Insect Molecular Biology, 2014

The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetlederived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect-or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle.

Diversity of Beetle Genes Encoding Novel Plant Cell Wall Degrading Enzymes

PLoS ONE, 2010

Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent ''disappearance'' of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology.