Quantification of symbiotic contributions to lower termite lignocellulose digestion using antimicrobial treatments

Animal-microbe co-evolution and symbiosis are broadly distributed across the animal kingdom. Insects form a myriad of associations with microbes ranging from vectoring of pathogens to intracellular, mutualistic relationships. Lower termites are key models for insect-microbe symbiosis because of the diversity, complexity and functionality of their unique tripartite symbiosis. This collaboration allows termites to live on a diet of nitrogen-poor lignocellulose. Recent functional investigations of lignocellulose digestion in lower termites have primarily focused on the contributions of the eukaryotic members of the termite holobiont (termite and protist). Here, using multiple antimicrobial treatments, we induced differing degrees of dysbiosis in the termite gut, leading to variably altered symbiont abundance and diversity, and lignocellulolytic capacity. Although protists are clearly affected by antimicrobial treatments, our findings provide novel evidence that the removal of distinct groups of bacteria partially reduces, but does not abolish, the saccharolytic potential of the termite gut holobiont. This is specifically manifested by reductions of 23–47% and 30–52% in glucose and xylose yields respectively from complex lignocellulose. Thus, all members of the lower termite holobiont (termite, protist and prokaryotes) are involved in the process of efficient, sustained lignocellulase activity. This unprecedented quantification of the relative importance of prokaryotes in this system emphasizes the collaborative nature of the termite holobiont, and the relevance of lower termites as models for inter-domain symbioses.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Influence of lignin in Reticulitermes santonensis: symbiotic interactions investigated through proteomics

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment.     

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.     

高效降解木质纤维素的白蚁肠道微生物组

木食性白蚁是自然界木质纤维素的高效降解者,在长期进化过程中白蚁与其肠道微生物组协同作用发展出不同的纤维素降解机制。木食性白蚁具有分别来源于白蚁和共生微生物的两套纤维素酶系统。在低等白蚁中,木质颗粒经过白蚁前、中肠分泌的内源性酶初步消化后,在后肠共生鞭毛虫中被降解为乙酸、二氧化碳和氢。高等木食性白蚁在进化中丢失了鞭毛虫,木质颗粒经白蚁自身分泌的酶初步消化后,在后肠大量共生细菌的帮助下被有效降解。培菌类白蚁利用其菌圃中的蚁巢伞菌和肠道微生物协同作用降解木质纤维素。共生微生物在白蚁的氮素固定与循环、中间产物代谢及纤维素降解等过程中发挥了重要作用。学习和模拟白蚁高效降解木质纤维素的体系,对生物质能源的产业化发展具有积极的意义。     

Aromatic compound degradation by the wood-feeding termite Coptotermes formosanus (Shiraki)

Wood-feeding termites (WFT) have proven to be highly efficient for wood digestion. There is evidence to support the hypothesis that there are ligninolytic enzymes existing in the gut of WFT responsible for wood pretreatment toward cellulose utilization. Elucidating the mechanism of biomass pretreatment through lignin modification in termites will help to develop more efficient lignocellulosic biofuel production processes. The in-vivo degradation of aromatic compounds with different substructures, including dyes, lignin model monomers and dimers, and lignin sulfonate, by Coptotermes formosanus (Shiraki) was investigated. The degradation of aromatic compounds was determined using pyrolysis-gas chromatography/mass spectrometry. The results revealed that WFT were able to metabolize the conjugated aromatic structures and that the degradation efficiency is higher in the foregut and midgut regions than in the hindgut. This is the first time that evidence has been provided to show different aromatic compound degradation in the separate gut segments of a termite. This study provides information on the C. formosanus (Shiraki) lignin modification phenomenon, and it demonstrates that phenomenon’s potential in the breakdown of the plant cell wall. Understanding this lignin modification could contribute to technology that will supplant current harsh pretreatment protocols for plant cell walls and thereby better facilitate the conversion of cellulose and hemicellulose.     

Potential of termite-based biomass pre-treatment strategies for use in bioethanol production

Abstract When considering the current state of the biorefinery industry, it is readily apparent that industrial cellulose and hemicellulose digestion processes are relatively advanced, whereas enzymatic pre-treatment strategies for biomass delignification and cellulose solubilization are not well developed. The need for efficient biomass pre-treatment strategies presents a significant opportunity for researchers studying lignocellulose digestion in termites and other insects. With an emphasis on industrial biomass pre-treatment, this review provides an overview of: (i) industrial biorefining operations (feedstocks, processing, and economics); (ii) recent findings from termite research that have revealed candidate enzymes; and (iii) research needs and opportunities for consideration by entomologists working in this area. With respect to research findings, recently identified candidate lignases (laccases, catalases, peroxidases, esterases), other potentially important detoxification enzymes (cytochrome P450, superoxide dismutase), and phenolic acid esterases (carboxylesterases) that may assist in hemicellulose solubilization are overviewed. Regarding research needs and opportunities, several approaches for identification of candidate pre-treatment enzymes from upstream, symbiont-free gut regions are also described.     

Dietary‐driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clément

The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose‐based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials. All non‐treated sound woods produced similar flagellate protist communities that were more diverse and of high evenness (low dominance). On the contrary, flagellate protist communities from cellulose‐fed termites and starving termites were considered to be significantly different from all non‐treated woods; they were less diverse and some morphotypes became dominant as a consequence of flagellate protist communities having suffered major adaptations to these diets. The flagellate protist communities of untreated beech and thermally modified beech‐fed termites were considered to be significantly different in terms of abundance and morphotype diversity. This may be caused by a decrease in lignocellulose quality available for termites and from an interference of thermally treated wood with the chemical stability of the termite hindgut. Our study suggests that as a consequence of the strong division of labour among these protists to accomplish the intricate process of lignocellulose digestion, termite symbiotic flagellate protist communities are a dynamic assemblage able to adapt to different conditions and diets. This study is important for the community‐level alteration approach, and it is the first study to investigate the effects of thermally modified wood on the flagellate protist communities of subterranean termites.     

The Enterobacterium Trabulsiella odontotermitis Presents Novel Adaptations Related to Its Association with Fungus-Growing Termites

Fungus-growing termites rely on symbiotic microorganisms to help break down plant material and to obtain nutrients. Their fungal cultivar, Termitomyces, is the main plant degrader and food source for the termites, while gut bacteria complement Termitomyces in the degradation of foodstuffs, fixation of nitrogen, and metabolism of amino acids and sugars. Due to the community complexity and because these typically anaerobic bacteria can rarely be cultured, little is known about the physiological capabilities of individual bacterial members of the gut communities and their associations with the termite host. The bacterium Trabulsiella odontotermitis is associated with fungus-growing termites, but this genus is generally understudied, with only two described species. Taking diverse approaches, we obtained a solid phylogenetic placement of T. odontotermitis among the Enterobacteriaceae, investigated the physiology and enzymatic profiles of T. odontotermitis isolates, determined the localization of the bacterium in the termite gut, compared draft genomes of two T. odontotermitis isolates to those of their close relatives, and examined the expression of genes relevant to host colonization and putative symbiont functions. Our findings support the hypothesis that T. odontotermitis is a facultative symbiont mainly located in the paunch compartment of the gut, with possible roles in carbohydrate metabolism and aflatoxin degradation, while displaying adaptations to association with the termite host, such as expressing genes for a type VI secretion system which has been demonstrated to assist bacterial competition, colonization, and survival within hosts.     

Pyrotag Sequencing of the Gut Microbiota of the Cockroach Shelfordella lateralis Reveals a Highly Dynamic Core but Only Limited Effects of Diet on Community Structure

Although blattid cockroaches and termites share a common ancestor, their diets are distinctly different. While termites consume a highly specialized diet of lignocellulose, cockroaches are omnivorous and opportunistic feeders. The role of the termite gut microbiota has been studied intensively, but little is known about the cockroach gut microbiota and its function in digestion and nutrition, particularly the adaptation to different diets. Our analyses of the bacterial gut microbiota of the blattid cockroach Shelfordella lateralis combining terminal restriction fragment length polymorphism of their 16S rRNA genes with physiological parameters (microbial metabolites, hydrogen and methane emission) indicated substantial variation between individuals but failed to identify any diet-related response. Subsequent deep-sequencing of the 16S rRNA genes of the colonic gut microbiota of S. lateralis fed either a high- or a low-fiber diet confirmed the absence of bacterial taxa that responded to diet. Instead, we found a small number of abundant phylotypes that were consistently present in all samples and made up half of the community in both diet groups. They varied strongly in abundance between individual samples at the genus but not at the family level. The remaining phylotypes were inconsistently present among replicate batches. Our findings suggest that S. lateralis harbors a highly dynamic core gut microbiota that is maintained even after fundamental dietary shifts, and that any dietary effects on the gut community are likely to be masked by strong individual variations.     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

不同饲料饲养的近暗散白蚁工蚁肠道中差异蛋白分析

【目的】本研究旨在通过比较不同木质纤维素含量的饲料对近暗散白蚁Reticulitermesperilucifugus工蚁前中肠和后肠内容物蛋白的组成和表达差异,为揭示白蚁的营养消化吸收机理提供蛋白水平上的依据。【方法】用木质纤维素含量不同的3种饲料(松木、秸秆和滤纸)饲养近暗散白蚁工蚁,然后用双向电泳分析肠道不同部位的内容物蛋白,并对差异蛋白进行MALDI-TOF/MS测序及生物信息学分析。【结果】双向电泳结果发现,同一饲料饲养的近暗散白蚁工蚁前中肠可辨蛋白点数明显多于后肠;不同饲料饲喂的工蚁前中肠和后肠可辨蛋白点数依次是松木饲养的＞滤纸饲养的＞秸秆饲养的。通过对115个蛋白点的测序分析表明,不同饲料饲喂的工蚁前中肠和后肠中的主要差异蛋白为具有催化活性的蛋白质,包括与氨基酸代谢、丙酮酸代谢、碳代谢、氮代谢、TCA循环、糖酵解、糖异生和纤维素降解等相关的酶,以及参与细胞组成和信号传导的蛋白。【结论】木质纤维素含量不同的饲料饲养的近暗散白蚁工蚁肠道中的差异蛋白主要为具有催化活性的蛋白、细胞组成蛋白和信号传导蛋白,表明不同的饲料影响近暗散白蚁的肠道蛋白组成。这些结果为揭示白蚁对木质纤维素的降解机理提供了参考。     

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut‐dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture‐ and cloning‐dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high‐throughput 454 pyrosequencing of 16S V5‐V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin‐rich and lignin‐poor cellulose diets after a 7‐day feeding period. Of the ~475 000 high‐quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species‐level phylotypes. However, diet‐dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony‐level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.