Phylogenetic Identification of the Symbiotic Hypermastigote Trichonympha agilis in the Hindgut of the Termite Reticulitermes speratus Based on Small-Subunit rRNA Sequence

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.     

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.     

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     

Phylogenetic Placement of Trichonympha

Flagellated protists of the Class Hypermastigida have previously been classified on morphology alone, since no molecular sequences have been available. We have isolated DNA from 350 cells of the hypermastigote Trichonympha , manually collected from the hindgut of Zootermopsis angusticollis , and used this DNA as template for polymerase chain reaction amplification of the small-subunit ribosomal RNA gene. The DNA sequence of the amplified fragment is closely related to that of a previously-unidentified gut symbiont from the termite Reticulitermes flavipes , and phylogenetic analysis places both sequences as a sister group to the known trichomonads. in agreement with the morphological classification.     

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.     

Phylogenetic Diversity of Nitrogen Fixation Genes in the Intestinal Tract of Reticulitermes chinensis Snyder

Wood-feeding termites live on cellulolytic materials that typically lack of nitrogen sources. It was reported that symbiotic microbes play important roles in the maintenance of a normal nitrogen contents in termite by different metabolisms including nitrogen fixation. In this study, the diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of Reticulitermes chinensis Snyder was investigated with culture independent method. Fragments of the nifH genes, which encode dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut with four sets of primers corresponding to the conserved regions of the genes. Clones were randomly selected and analyzed by RFLP. Sequence analysis revealed that a large number of nifH sequences retrieved from the termite gut were most closely related to strict anaerobic bacteria such as clostridia and spirochetes, some of the others were affiliated with proteobacteria, bacteroides, or methanogenic archaea. The results showed that there was a remarkable diversity of nitrogenase genes in the gut of Reticulitermes chinensis Snyder.     

“Endomicrobia”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of Reticulitermes santonensis belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of Reticulitermes speratus that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus “Endomicrobium.” Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name “Endomicrobia,” are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.     

Cospeciation of termite gut flagellates and their bacterial endosymbionts: Trichonympha species and 'Candidatus Endomicrobium trichonymphae'

Symbiotic flagellates play a major role in the digestion of lignocellulose in the hindgut of lower termites. Many termite gut flagellates harbour a distinct lineage of bacterial endosymbionts, so-called Endomicrobia, which belong to the candidate phylum Termite Group 1. Using an rRNA-based approach, we investigated the phylogeny of Trichonympha , the predominant flagellates in a wide range of termite species, and of their Endomicrobia symbionts. We found that Trichonympha species constitute three well-supported clusters in the Parabasalia tree. Endomicrobia were detected only in the apical lineage (Cluster I), which comprises flagellates present in the termite families Termopsidae and Rhinotermitidae, but apparently absent in the basal lineages (Clusters II and III) consisting of flagellates from other termite families and from the wood-feeding cockroach, Cryptocercus punctulatus . The endosymbionts of Cluster I form a monophyletic group distinct from many other lineages of Endomicrobia and seem to have cospeciated with their flagellate host. The distribution pattern of the symbiotic pairs among different termite species indicates that cospeciation of flagellates and endosymbionts is not simply the result of a spatial separation of the flagellate lineages in different termite species, but that Endomicrobia are inherited among Trichonympha species by vertical transmission. We suggest extending the previously proposed candidatus name ' Endomicrobium trichonymphae ' to all Endomicrobia symbionts of Trichonympha species, and estimate that the acquisition by an ancestor of Trichonympha Cluster I must have occurred about 40–70 million years ago, long after the flagellates entered the termites.     

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.     

Effect of Temperature and Termite Starvation on Phagocytosis by Protozoan Symbionts of the Eastern Subterranean Termite Reticulitermes flavipes Kollar

Abstract Many termite species rely on intestinal protozoan symbionts to digest their cellulosic foods. We examined cellulose acquisition by the symbionts of the Eastern subterranean termite Reticulitermes flavipes Kollar (Isoptera; Rhinotermitidae) by following their phagocytosis of red paper fed to the termite host. The effects of termite host starvation and environmental temperature on feeding activity were studied in the zooflagellates Trichonympha agilis Leidy (Trichonymphidae), Pyrsonympha vertens Leidy, Dinenympha fimbriata Kirby, and D. gracilis Leidy (Pyrsonymphidae), which are among the largest residents in R. flavipes' hindguts. Protozoans in termites starved for 24 h ingested red paper significantly sooner than protozoans in termites with continuous access to food. Trichonympha, Pyrsonympha, and Dinenympha all ingested red paper particles at approximately the same rate. Red paper appeared significantly sooner in protozoans in termites maintained at 32°C than in those maintained at 22°C or 26°C. At 32°C, numbers of Trichonympha per gut remained constant over 96 h. Pyrsonympha and Dinenympha cells were absent or significantly reduced in number by 72 h at that temperature. These results provide insight into the environmental factors that shape the termite–protozoan symbiosis. They may aid in the development of protozoicides used to control pest termites. Received: 1 August 1997; Accepted: 26 November 1997     

Molecular Characterization of Parabasalian Symbionts Coronympha clevelandii and Trichonympha subquasilla from the Hawaiian Lowland Tree Termite Incisitermes immigrans

An important and undervalued challenge in characterizing symbiotic protists is the accurate identification of their host species. Here, we use DNA barcoding to resolve one confusing case involving parabasalian symbionts in the hindgut of the Hawaiian lowland tree termite, Incisitermes immigrans, which is host to several parabasalians, including the type species for the genus Coronympha, C. clevelandii. We collected I. immigrans from its type locality (Hawaii), confirmed its identity by DNA barcoding, and characterized the phylogenetic position of two symbionts, C. clevelandii and Trichonympha subquasilla. These data show that previous molecular surveys of “I. immigrans” are, in fact, mainly derived from the Caribbean termite I. schwarzi, and perhaps also another related species. These results emphasize the need for host barcoding, clarify the relationship between morphologically distinct Coronympha species, and also suggest some interesting distribution patterns of nonendemic termite species and their symbionts.     

Isolation and characterization of enteric bacteria from the hindgut of Formosan termite

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is an aggressive, invasive termite species that has caused billions of dollars of damage across the United States for the past 50 years. Termites depend on intestinal microorganisms for cellulose digestion. Symbiotic microorganisms in the termite gut play key physiological functions such as cellulose and hemicellulose digestion, acetogenesis, hydrogenesis, methanogenesis, sulfate reduction, and nitrogen fixation. Additionally, intestinal microbes create suitable conditions for symbiotic protozoans through the production of nutrients and the maintenance of the pH and the anaerobic conditions in the gut. Although extensive research has been done on the symbiotic relationship of these termites and the microbes found in its gut, there is little information available on the role of facultative anaerobes in the gut. We isolated four enteric bacteria from the hindgut of Formosan subterranean termite, C. formosanus. All isolates were facultative anaerobes and G-. The isolates were identified as Serratia marcescens, Enterobacter aerogens, Enterobacter cloacae, and Citrobacter farmeri by using BIOLOG assay and fatty acid methyl ester analysis (FAME). Each isolate was characterized using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and biochemical study. This is the first report on the presence of facultative microbes in termite gut. Results of this first study on facultative microbes in the termite gut indicate that the role of facultative organisms in the Formosan termite gut may be to scavenge oxygen and create anaerobic conditions for the anaerobic microorganisms, which are essential for digestion of cellulose consumed by the termite.     

Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes

The symbiotic digestion of lignocellulose in the hindgut of the wood-feeding termite Reticulitermes flavipes is characterized by two major metabolic pathways: (i) the oxidation of polysaccharides to acetate by anaerobic hydrogen-producing protozoa; and (ii) the reduction of CO2 by hydrogenotrophic acetogenic bacteria. Both reactions together would render the hindgut largely homoacetogenic. However, the results of this study show that the situation is more complex. By microinjection of radiolabelled metabolites into intact agarose-embedded hindguts, we showed that the in situ rates of reductive acetogenesis (3.3 nmol termite(-1) h(-1)) represent only 10% of the total carbon flux in the living termite, whereas 30% of the carbon flux proceeds via lactate. The rapid turnover of the lactate pool (7.2 nmol termite(-1) h(-1)) consolidates the previously reported presence of lactic acid bacteria in the R. flavipes hindgut and the low lactate concentrations in the hindgut fluid. However, the immediate precursor of lactate remains unknown; the low turnover rates of injected glucose (< 0.5 nmol termite(-1) h(-1)) indicate that free glucose is not an important intermediate under in situ conditions. The influence of the incubation atmosphere on the turnover rate and the product pattern of glucose and lactate confirmed that the influx of oxygen via the gut epithelium and its reduction in the hindgut periphery have a significant impact on carbon and electron flow within the hindgut microbial community. The in situ rates of reductive acetogenesis were not significantly affected by the presence of oxygen or exogenous H2, which is in agreement with a localization of homoacetogens in the anoxic gut lumen rather than in the oxic periphery. This adds strong support to the hypothesis that the co-existence of methanogens and homoacetogens in this termite is based on the spatial arrangement of the different populations of the gut microbiota. A refined model of metabolic fluxes in the hindgut of R. flavipes is presented.     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

‘Candidatus Ancillula trichonymphae’, a novel lineage of endosymbiotic Actinobacteria in termite gut flagellates of the genus Trichonympha

Termite gut flagellates are colonized by host‐specific lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluorescence in situ hybridization and electron microscopy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonympha species from different termites. The flagellates in Trichonympha Cluster I were the only species associated with ‘Endomicrobia’, which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked ‘Endomicrobia’ and were associated with endosymbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name ‘Candidatus Ancillula trichonymphae’, represent a novel, deep‐branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Trichonympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplasmic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invaginations of the plasma membrane that were clearly connected to the exterior of the host cell.     

Effects of antibiotics on hydrogen production and gut symbionts in the Formosan subterranean termite Coptotermes formosanus (Isoptera: Rhinotermitidae)

Abstract Symbiotic microorganisms that inhabit the gut of Coptotermes formosanus enable this termite to degrade lignocelluloses and further produce hydrogen as an important intermediate to be recycled in its hindgut or as a byproduct to be emitted to the atmosphere. Both symbiotic protists and prokaryotes in the guts of termites demonstrated some different roles with respect to hydrogen production. In this study, the effects of two antibiotics, ampicillin and tetracycline, on hydrogen emission and the gut symbionts of C. formosanus were investigated. Hydrogen emission from termite guts was significantly enhanced when termites fed on wood diets treated with either ampicillin or tetracycline. The greatest H₂ emission rates, 2 519 ± 74 and 2 080 ± 377 nmol/h/g body weight, were recorded with the treatments of ampicillin and tetracycline, respectively, which showed 6–7 times more H₂ production than that of controls. Antibiotic‐treated diets negatively affected the prokaryotic communities and reduced their abundances, particularly on those ectosymbionts inhabiting the gut walls or in the gut fluid of C. formosanus, such as spirochetes. However, no significant reductions in the counts of gut cellulolytic protists, Pseudotrichonympha grassii and Holomastigotoids hartmanni, were recorded; and with a further observation by confocal laser scanning microscopy, the endosymbionts inhabiting P. grassii generally survived the antibiotic treatments. These results suggest that some prokaryotes may serve as the main hydrogen consumers, while P. grassii, together with its endosymbionts, may function as the main contributors for hydrogen production in the hindgut of C. formosanus.