Symbiotic flagellate protists as cryptic drivers of adaptation and invasiveness of the subterranean termite Reticulitermes grassei Clément

Changes in flagellate protist communities of subterranean termite Reticulitermes grassei across different locations were evaluated following four predictions: (i) Rural endemic (Portugal mainland) termite populations will exhibit high diversity of symbionts; (ii) invasive urban populations (Horta city, Faial island, Azores), on the contrary, will exhibit lower diversity of symbionts, showing high similarity of symbiont assemblages through environmental filtering; (iii) recent historical colonization of isolated regions—as the case of islands—will imply a loss of symbiont diversity; and (iv) island isolation will trigger a change in colony breeding structure toward a less aggressive behavior. Symbiont flagellate protist communities were morphologically identified, and species richness and relative abundances, as well as biodiversity indices, were used to compare symbiotic communities in colonies from urban and rural environments and between island invasive and mainland endemic populations. To evaluate prediction on the impact of isolation (iv), aggression tests were performed among termites comprising island invasive and mainland endemic populations. A core group of flagellates and secondary facultative symbionts was identified. Termites from rural environments showed, in the majority of observed colonies, more diverse and abundant protist communities, probably confirming prediction (i). Corroborating prediction (ii), the two least diverse communities belong to termites captured inside urban areas. The Azorean invasive termite colonies had more diverse protist communities than expected and prediction (iii) which was not verified within this study. Termites from mainland populations showed a high level of aggressiveness between neighboring colonies, in contrast to the invasive colonies from Horta city, which were not aggressive to neighbors according to prediction (iv). The symbiotic flagellate community of R. grassei showed the ability to change in a way that might be consistent with adaptation to available conditions, possibly contributing to optimization of the colonization of new habitats and spreading of its distribution area, highlighting R. grassei potential as an invasive species.     

Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose

Wood‐feeding lower termites harbour symbiotic gut protists that support the termite nutritionally by degrading recalcitrant lignocellulose. These protists themselves host specific endo‐ and ectosymbiotic bacteria, functions of which remain largely unknown. Here, we present draft genomes of a dominant, uncultured ectosymbiont belonging to the order Bacteroidales, ‘Candidatus Symbiothrix dinenymphae’, which colonizes the cell surface of the cellulolytic gut protists Dinenympha spp. We analysed four single‐cell genomes of Ca. S. dinenymphae, the highest genome completeness was estimated to be 81.6–82.3% with a predicted genome size of 4.28–4.31 Mb. The genome retains genes encoding large parts of the amino acid, cofactor and nucleotide biosynthetic pathways. In addition, the genome contains genes encoding various glycoside hydrolases such as endoglucanases and hemicellulases. The genome indicates that Ca. S. dinenymphae ferments lignocellulose‐derived monosaccharides to acetate, a major carbon and energy source of the host termite. We suggest that the ectosymbiont digests lignocellulose and provides nutrients to the host termites, and hypothesize that the hydrolytic activity might also function as a pretreatment for the host protist to effectively decompose the crystalline cellulose components.     

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.     

Possible evolutionary significance of spirochaetes.

Large symbiotic spirochaetes of the family Pillotaceae (e.g. pillotinas) are found in dry wood and subterranean termites (Hollande & Garagozlou 1967). These morphologically distinctive spirochaetes comprise several genera. Some of them contain microtubules within their protoplasmic cylinders. They demonstrate a variety of relations with their termite and protist hosts. Some are free-living within the lumen of the intestine, some tend to be associated with filamentous and other bacteria, some are found regularly coursing between the numerous undulipodia ( = eukaryotic flagella, cilia, and other (9 + 2) organelles of motility) of hypermastigotes and polymastigotes. Still other smaller termite spirochaetes are regularly attached to protists via specialized attachment sites. Some even form motility symbiosis with their host protists. The analogy between the behaviour of host-associated spirochaetes and the possible steps in the origin of the undulipodia and mitotic system of eukaryotes is discussed briefly.     

白蚁及共生微生物木质纤维素水解酶的种类

白蚁是热带生态系统重要的木质纤维素降解者。白蚁种类丰富,可分成高等白蚁和低等白蚁,食性也具有各自特点。白蚁自身可以产生纤维素酶,主要是GHF9的内切葡聚糖酶(EG),也有β-葡萄糖苷酶(GB)。低等白蚁共生的原虫中已发现丰富的纤维素酶基因,属于GHF5,7和45。同时还有其他相关功能基因,如木聚糖酶和果胶类物质水解酶。高等白蚁肠道中没有共生原虫。高等培菌白蚁可以利用共生蚁巢伞属真菌促进木质纤维素降解,真菌可以产生纤维素酶,果胶质水解酶类、木聚糖酶,同时还产生可能与木质素分解相关的一种漆酶,但是从分子水平,关于共生真菌纤维素水解酶的研究还较少。白蚁肠道已分离出许多具有木质纤维素降解能力的菌株,最近的研究也发现了大量细菌纤维素酶基因。白蚁-共生系统丰富的木质纤维素水解酶类为发展生物方法开发纤维素乙醇这一思路提供有价值的资源。     

Phylogenetic Analysis of Cellulolytic Enzyme Genes from Representative Lineages of Termites and a Related Cockroach

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.     

Diversity and resilience of the wood‐feeding higher termite Mironasutitermes shangchengensis gut microbiota in response to temporal and diet variations

Termites are considered among the most efficient bioreactors, with high capacities for lignocellulose degradation and utilization. Recently, several studies have characterized the gut microbiota of diverse termites. However, the temporal dynamics of the gut microbiota within a given termite with dietary diversity are poorly understood. Here, we employed 16S rDNA barcoded pyrosequencing analysis to investigate temporal changes in bacterial diversity and richness of the gut microbiota of wood‐feeding higher termite Mironasutitermes shangchengensis under three lignocellulose content‐based diets that feature wood, corn stalks, and filter paper. Compositions of the predominant termite gut residents were largely constant among the gut microbiomes under different diets, but each diet caused specific changes in the bacterial composition over time. Notably, microbial communities exhibited an unexpectedly strong resilience during continuous feeding on both corn stalks and filter paper. Members of five bacterial phyla, that is, Spirochaetes, Firmicutes, Actinobacteria, Tenericutes, and Acidobacteria, were strongly associated with the resilience. These findings provide insights into the stability of the gut microbiota in higher termites and have important implications for the future design of robust bioreactors for lignocellulose degradation and utilization.     

The GH26 β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: Heterologous expression and characterization

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.     

肠道鞭毛虫群落及在宿主白蚁分类学上的意义

于国内五个常见白蚁种肠道内共记录到十七个鞭毛虫种。鞭毛虫种群具特异性和稳定性。群落相的变异度反映了宿主白蚁亲缘关系的离散度。异域同种白蚁肠道鞭毛虫群落显示稳定的同一相,同属异种白蚁的鞭毛虫群落部分相同;异科异属或同科异属白蚁的鞭毛虫群落绝然相异。 鉴于传统的生物分类法已明显地显示出其片面性及局限性,为此,人们正试图从其它学科领域,各个不同角度去探索能更客观反映各物种在系统发生上关系的新分类法。迄今,精确定性的生化分类、免疫分类和以群体力对象的定量的数量分类等已倪露头角,从而为以形态描述为主的经典生物分类学注入了新的活力。 本文试图通过对白蚁肠道鞭毛虫群落及种群的研究而借以探讨其在宿主白蚁系统分类研究中的潜在意义。     

Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.     

Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis

Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-β-1,4-glucanase and β-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-β-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.     

Phylogenetic identification of symbiotic protists of five Chinese Reticulitermes species indicates a cospeciation of gut microfauna with host termites

Symbiotic protists play important roles in the wood digestion of lower termites. Previous studies showed that termites generally possess host-specific flagellate communities. The genus Reticulitermes is particularly interesting because its unique assemblage of gut flagellates bears evidence for transfaunation. The gut fauna of Reticulitermes species in Japan, Europe, and North America had been investigated, but data on species in China are scarce. For the first time, we analyzed the phylogeny of protists in the hindgut of five Reticulitermes species in China. A total of 22 protist phylotypes were affiliated with the family Trichonymphidae, Teranymphidae, Trichomonadidae, and Holomastigotoididae (Phylum Parabasalia), and 45 protist phylotypes were affiliated with the family Pyrsonymphidae (Phylum Preaxostyla). The protist fauna of these five Reticulitermes species is similar to those of Reticulitermes species in other geographical regions. The topology of Trichonymphidae subtree was similar to that of Reticulitermes tree. All Preaxostyla clones were affiliated with the genera Pyrsonympha and Dinenympha (Order Oxymonadida) as in the other Reticulitermes species. The results of this study not only add to the existing information on the flagellates present in other Reticulitermes species but also offer the opportunity to test the hypotheses for the coevolution of symbiotic protists with their host termites.     

Complex coevolutionary history of symbiotic Bacteroidales bacteria of various protists in the gut of termites

Background  

The microbial community in the gut of termites is responsible for the efficient decomposition of recalcitrant lignocellulose. Prominent features of this community are its complexity and the associations of prokaryotes with the cells of cellulolytic flagellated protists. Bacteria in the order Bacteroidales are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. In particular, ectosymbionts exhibit distinct morphological patterns of the associations. Therefore, these Bacteroidales symbionts provide an opportunity to investigate not only the coevolutionary relationships with the host protists and their morphological evolution but also how symbiotic associations between prokaryotes and eukaryotes occur and evolve within a complex symbiotic community.     

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.     

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。     

低等白蚁肠道共生微生物的多样性及其功能

低等白蚁肠道里存在着复杂的微生物区系,包括真核微生物鞭毛虫和原核生物,细菌及古细菌。低等白蚁的后肠以特别膨大的囊形胃及其氢氧浓度的明显梯度分布和丰富的微生物区系为特征,是白蚁进行木质纤维素消化的主要器官。后肠内的鞭毛虫能将纤维素水解并发酵为乙酸,二氧化碳和氢,为白蚁提供营养和能源。系统发育研究表明,低等白蚁肠道共生细菌的主要类群为白蚁菌群1、螺旋体、拟杆菌,低G C mol%含量的革兰氏阳性菌和紫细菌等。而古细菌主要为甲烷短杆菌属的产甲烷菌。共生原核生物与二氧化碳的还原和氮的循环等代谢有关。但肠道共生微生物的具体功能和作用机制还有待进一步的揭示。     

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.