Molecular phylogeny of symbiotic basidiomycetes of fungus-growing termites in Thailand and their relationship with the host

Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi with host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.     

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.     

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.     

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H(2) production in termite guts has been largely underestimated.     

The evolution of the flagellar assembly pathway in endosymbiotic bacterial genomes

Genome shrinkage is a common feature of most intracellular pathogens and symbionts. Reduction of genome sizes is among the best-characterized evolutionary ways of intracellular organisms to save and avoid maintaining expensive redundant biological processes. Endosymbiotic bacteria of insects are examples of biological economy taken to completion because their genomes are dramatically reduced. These bacteria are nonmotile, and their biochemical processes are intimately related to those of their host. Because of this relationship, many of the processes in these bacteria have been either lost or have suffered massive remodeling to adapt to the intracellular symbiotic lifestyle. An example of such changes is the flagellum structure that is essential for bacterial motility and infectivity. Our analysis indicates that genes responsible for flagellar assembly have been partially or totally lost in most intracellular symbionts of gamma-Proteobacteria. Comparative genomic analyses show that flagellar genes have been differentially lost in endosymbiotic bacteria of insects. Only proteins involved in protein export within the flagella assembly pathway (type III secretion system and the basal body) have been kept in most of the endosymbionts, whereas those involved in building the filament and hook of flagella have only in few instances been kept, indicating a change in the functional purpose of this pathway. In some endosymbionts, genes controlling protein-export switch and hook length have undergone functional divergence as shown through an analysis of their evolutionary dynamics. Based on our results, we suggest that genes of flagellum have diverged functionally as to specialize in the export of proteins from the bacterium to the host.     

Evidence for cocladogenesis between diverse dictyopteran lineages and their intracellular endosymbionts

Bacteria of the genus Blattabacterium are intracellular symbionts that reside in specialized cells of cockroaches and the termite Mastotermes darwiniensis. They appear to be obligate mutualists, and are transmitted vertically in the eggs. Such characteristics are expected to lead to equivalent phylogenies for host and symbiont, and we tested this hypothesis using recently accumulated data on relationships among termites and cockroaches and their Blattabacterium spp. Host and symbiont topologies were found to be highly similar, and various tests indicated that they were not statistically different. A close relationship between endosymbionts from termites and members of the wood-feeding cockroach genus Cryptocercus was found, supporting the hypothesis that the former evolved from subsocial, wood-dwelling cockroaches. The majority of the Blattabacterium spp. sequences appear to have undergone similar rates of evolution since their divergence from a common ancestor, and an estimate of this rate was determined based on early Cretaceous host fossils. The results support the idea that the stem group of modern cockroaches radiated sometime between the late Jurassic and early Cretaceous-not the Carboniferous, as has been suggested on the basis of roach-like fossils from this epoch.     

Endosymbiotic Bacteroidales bacteria of the flagellated protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

Bacterial Endosymbiosis in a Chordate Host: Long-Term Co-Evolution and Conservation of Secondary Metabolism

Intracellular symbiosis is known to be widespread in insects, but there are few described examples in other types of host. These symbionts carry out useful activities such as synthesizing nutrients and conferring resistance against adverse events such as parasitism. Such symbionts persist through host speciation events, being passed down through vertical transmission. Due to various evolutionary forces, symbionts go through a process of genome reduction, eventually resulting in tiny genomes where only those genes essential to immediate survival and those beneficial to the host remain. In the marine environment, invertebrates such as tunicates are known to harbor complex microbiomes implicated in the production of natural products that are toxic and probably serve a defensive function. Here, we show that the intracellular symbiont Candidatus Endolissoclinum faulkneri is a long-standing symbiont of the tunicate Lissoclinum patella, that has persisted through cryptic speciation of the host. In contrast to the known examples of insect symbionts, which tend to be either relatively recent or ancient relationships, the genome of Ca. E. faulkneri has a very low coding density but very few recognizable pseudogenes. The almost complete degradation of intergenic regions and stable gene inventory of extant strains of Ca. E. faulkneri show that further degradation and deletion is happening very slowly. This is a novel stage of genome reduction and provides insight into how tiny genomes are formed. The ptz pathway, which produces the defensive patellazoles, is shown to date to before the divergence of Ca. E. faulkneri strains, reinforcing its importance in this symbiotic relationship. Lastly, as in insects we show that stable symbionts can be lost, as we describe an L. patella animal where Ca. E. faulkneri is displaced by a likely intracellular pathogen. Our results suggest that intracellular symbionts may be an important source of ecologically significant natural products in animals.     

Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and parabasalian symbionts in the hindgut of termites

The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.     

白蚁共生放线菌研究进展

白蚁与微生物的共生关系是目前较受关注的研究热点,其肠道及巢内的共生微生物在降解木质纤维素的过程中扮演着重要的角色。放线菌是这些共生微生物中的重要一类,广泛存在于肠道、蚁巢及其周围土壤中,目前已探明共生放线菌在参与白蚁碳氮循环及保护巢群免受外来病菌侵染等方面发挥着极大的作用。近年来,人们利用分子生物学技术鉴定了部分共生放线菌的类群,发现了许多具应用前景的新放线菌及相关酶和代谢产物。因此,研究与白蚁相关的放线菌不仅有助于人们了解白蚁共生菌群落间的互作及其与宿主间的关系,而且对人类开发自然资源也有较大的帮助。本文对白蚁共生放线菌的研究进展作一综述,供同行参考。     

The Role of Host Phylogeny Varies in Shaping Microbial Diversity in the Hindguts of Lower Termites

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.     

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).     

Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut

In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae'' and ‘Candidatus Nucleococcus kirbyi''. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Multipartite symbioses in fungus-growing termites (Blattodea: Termitidae,Macrotermitinae) for the degradation of lignocellulose

Fungus-growing termites are among the most successful herbivorous animals and improve crop productivity and soil fertility. A range of symbiotic organisms can be found inside their nests. However, interactions of termites with these symbionts are poorly understood. This review provides detailed information on the role of multipartite symbioses (between termitophiles, termites, fungi, and bacteria) in fungus-growing termites for lignocellulose degradation. The specific functions of each component in the symbiotic system are also discussed. Based on previous studies, we argue that the enzymatic contribution from the host, fungus, and bacteria greatly facilitates the decomposition of complex polysaccharide plant materials. The host–termitophile interaction protects the termite nest from natural enemies and maintains the stability of the microenvironment inside the colony.     

Multiple microalgal partners in symbiosis with the acantharian Acanthochiasma sp. (Radiolaria)

Acantharia (Radiolaria) are widespread and abundant heterotrophic marine protists, some of which can host endosymbiotic eukaryotic microalgae. Although this photosymbiotic association was first described at the end of the 19th century, the diversity of the symbiotic microalgae remains poorly characterized. Here, we examined the identity of the microalgae associated with the acantharian species Acanthochiasma sp. by sequencing partial 18S and internal transcribed spacer (ITS) ribosomal DNA genes from cultured symbionts and directly from isolated holobiont specimens. Single Acanthochiasma cells contained multiple symbiotic partners, including distantly related dinoflagellates (Heterocapsa sp., Pelagodinium sp., Azadinium sp. and Scrippsiella sp.) as well as a haptophyte (Chrysochromulina sp.). This original association of multiple symbiotic microalgae within a single host cell raises questions about the specificity and functioning of the relationship. These microalgae exhibit the common ecological feature of being abundant and widely distributed in coastal and oceanic waters, some occasionally forming extensive blooms. Some of the microalgal genera found in association with Acanthochiasma (i.e. Pelagodinium and Chrysochromulina) are known to occur in symbiosis with other heterotrophic protists such as Foraminifera and other Radiolaria, whereas Heterocapsa, Scrippsiella and Azadinium have never previously been reported to be involved in putative symbiotic relationships. The unusual association unveiled in this study contributes to our understanding of the ecological and evolutionary significance of photosymbiosis in Acantharia and also provides new insights into the nature of such partnerships in the planktonic realm.     

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218–221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences. Received: July 10, 2000 / Accepted: August 15, 2000     

Tsetse--A haven for microorganisms

Arthropods are involved in the transmission of parasitic and viral agents that cause devastating diseases in animals and plants. Effective control strategies for many of these diseases still rely on the elimination or reduction of vector insect populations. In addition to these pathogenic organisms, arthropods are rich in microbes that are symbiotic in their associations and are often necessary for the fecundity and viability of their hosts. Because the viability of the host often depends on these obligate symbionts, and because these organisms often live in close proximity to disease-causing pathogens, they have been of interest to applied biologists as a potential means to genetically manipulate populations of pest species. As knowledge on these symbiotic associations accumulates from distantly related insect taxa, conserved mechanisms for their transmission and evolutionary histories are beginning to emerge. Here, Serap Aksoy summarizes current knowledge on the functional and evolutionary biology of the multiple symbionts harbored in the medically and agriculturally important insect group, tsetse, and their potential role in the control of trypanosomiasis.     

昆虫共生微生物在病虫害和疾病控制上的应用前景

昆虫与微生物之间的互利共生关系是自然界中一种常见的互作形式。昆虫的种类丰富多样并且在自然界中分布广泛,在一定程度上得益于共生微生物的帮助。随着生物技术的不断发展,越来越多的共生微生物和互利共生模式得以发现并深入研究。微生物不仅能够为昆虫的生长发育提供营养,还能合成很多生物活性物质、调节宿主的免疫、对抗捕食者和抵御病原微生物感染,成为宿主昆虫健康和适应的守护者。鉴于共生微生物与昆虫生理生态的密切联系,以及昆虫对人类经济与健康的重要影响,利用共生微生物对昆虫及虫媒病进行生物控制已经成为一个热点研究方向,并展现了良好的应用前景。本文对昆虫共生微生物的多样性、生物学功能、与宿主相互作用机制及其在病虫害和虫媒病防治中的研究进展进行综述和展望。