Nitrogen mineralization, denitrification, and nitrate ammonification by soil-feeding termites: a 15N-based approach

Soil-feeding termites are abundant and play important roles in the biogeochemical processes in tropical soils. Previous studies indicated that they preferentially utilize the peptidic components of soil organic matter as a nutrient resource. Here, we determined the corresponding mineralization fluxes and elucidated other N transformation processes that occur during soil gut passage using ¹⁵N tracer techniques. Termite-based rates of N mineralization by Cubitermes umbratus and Cubitermes ugandensis in soil microcosms amended with ¹⁵NH₄ ⁺ were 6.6 and 9.2 nmol N day^?1 (g fresh wt)^?1, which means that the soil peptides fuel about 20 and 40% of the respiratory activity of these insects. Considering the areal biomass of soil-feeding termites in humid savannahs, soil-feeding termites should mineralize about 3% of the total N in their food soil per year. In addition to producing ammonia from ingested ¹⁵NO₃ ^? at approximately 10% of the mineralization rate, C. umbratus also formed N₂ at similar rates. The formation of labelled N₂ in microcosms amended with ¹⁵NH₄ ⁺ seems to be at least partially due to nitrification activity in the soil; evidence for the formation of nitrate in the posterior hindgut remains inconclusive. However, the so far unexplained increase of ¹⁵N abundance in the ammonia pools of the posterior hindgut compartments manifests additional hitherto unknown metabolic processes in this gut region. Collectively, our results not only reinforce the concept of nitrogenous soil components as an important dietary resource for soil-feeding termites, but also allow us to predict that N mineralization and nitrate ammonification activities in the termite gut should positively affect the dynamics of N in tropical soil.     

Nitrogen Mineralization, Ammonia Accumulation, and Emission of Gaseous NH3 by Soil-feeding Termites

There are numerous reports on the accumulation of ammonia in the mounds of soil-feeding termites. Here, we provided direct evidence for an effective mineralization of nitrogenous soil organic matter in the gut of Cubitermes spp., which gives rise to enormous ammonia concentrations in the intestinal tract. In Cubitermes ugandensis, the ammonia content of the nest material [24.5 μmol (g dry wt.)⁻¹] was about 300-fold higher than that of the parent soil. Large amounts of ammonia were present throughout the intestinal tract, with lowest values in the extremely alkaline gut sections (pH >12) and highest values posterior hindgut [185 μmol (g dry wt.)⁻¹]. Results obtained with other Cubitermes species were similar. Ammonia concentrations in the posterior hindgut of these humivorous species (up to 130 mM) are among the highest values ever reported for soil macroinvertebrates and are matched only by insects feeding on an extremely protein-rich diet (e.g., the sarcophageous larvae of blowflies). Volatilization of ammonia [about 10 nmol (g fresh wt.)⁻¹ h⁻¹], either directly by emission from the termite body or indirectly from their feces, led to NH₃ concentrations in the nest atmosphere of C. ugandensis that were three orders of magnitude above the ambient background – a relative accumulation that is considerably higher than that observed with CH₄ and CO₂. Together with previous results, these observations document that through their feeding activity and due to the physicochemical and biochemical properties of their digestive system, soil-feeding termites effectively catalyze the transformation of refractory soil organic nitrogen to a plant-available form that is protected from leaching by adsorption to the nest soil. Nitrogen mineralization rates of soil-feeding termites may surpass those effected by tropical earthworms and should contribute significantly to nitrogen fluxes in tropical ecosystems.     

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.     

High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.)

Higher termites are characterized by a purely prokaryotic gut microbiota and an increased compartmentation of their intestinal tract. In soil-feeding species, each gut compartment has different physicochemical conditions and is colonized by a specific microbial community. Although considerable information has accumulated also for wood-feeding species of the genus Nasutitermes, including cellulase activities and metagenomic data, a comprehensive study linking physicochemical gut conditions with the structure of the microbial communities in the different gut compartments is lacking. In this study, we measured high-resolution profiles of H(2), O(2), pH, and redox potential in the gut of Nasutitermes corniger termites, determined the fermentation products accumulating in the individual gut compartments, and analyzed the bacterial communities in detail by pyrotag sequencing of the V3-V4 region of the 16S rRNA genes. The dilated hindgut paunch (P3 compartment) was the only anoxic gut region, showed the highest density of bacteria, and accumulated H(2) to high partial pressures (up to 12 kPa). Molecular hydrogen is apparently produced by a dense community of Spirochaetes and Fibrobacteres, which also dominate the gut of other Nasutitermes species. All other compartments, such as the alkaline P1 compartment (average pH, 10.0), showed high redox potentials and comprised small but distinct populations characteristic for each gut region. In the crop and the posterior hindgut compartments, the community was even more diverse than in the paunch. Similarities in the communities of the posterior hindgut and crop suggested that proctodeal trophallaxis or coprophagy also occurs in higher termites. The large sampling depths of pyrotag sequencing in combination with the determination of important physicochemical parameters allow cautious conclusions concerning the functions of particular bacterial lineages in the respective gut sections to be drawn.     

Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.)

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.     

Axial Dynamics, Stability, and Interspecies Similarity of Bacterial Community Structure in the Highly Compartmentalized Gut of Soil-Feeding Termites (Cubitermes spp.)

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.     

Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.)

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (TERMITIDAE: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.     

Nitrate reduction to ammonia: a dissimilatory process in Enterobacter amnigenus

Thirty-four bacterial isolates from an agricultural soil anaerobically preincubated in the presence of glucose were tested for their ability to reduce nitrate to ammonia or to denitrify in two different media: nitrate broth and a minimal medium enriched with glucose. Ten isolates were considered denitrifying bacteria and 7 were dissimilatory ammonia producers. Ammonia production by the isolate identified as Enterobacter amnigenus was quantified and attained 50% of 138?mg?L(-1) of added NO(3)(-) N. The dissimilatory character of this reduction was clearly confirmed by culturing this (15)N-labeled bacterium in the presence of unlabeled nitrite. Nitrous oxide was produced at the same time as nitrite was reduced to ammonia. Increasing nitrate N levels from 48 to 553?mg?L(-1) in culture medium resulted in an increase in the level of nitrite produced and simultaneously a decrease in ammonia and nitrous oxide production. Key words: dissimilatory nitrate reduction, dissimilatory ammonia production, denitrification, Enterobacter amnigenus, (15)N.     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Nitrous oxide formation in the Colne estuary, England: the central role of nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N(2)O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N(2)O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N(2)O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 microM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 micromol N.m(-2).h(-1) with nitrite but only 180 micromol N.m(-2).h(-1) with nitrate. The ratios of rates of nitrous oxide production and denitrification (N(2)O/N(2) x 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N(2) formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N(2) via N(2)O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N(2)O as the end product. Consideration of free-energy changes during N(2)O formation led to the conclusion that N(2)O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N(2)O in high-nutrient-load estuaries.     

Differences between bacterial communities in the gut of a soil-feeding termite (Cubitermes niokoloensis) and its mounds

In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics.     

Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp.)

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (Termitidae: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.     

Isolation of facultatively aerobic actinomycetes from the gut, parent soil and mound materials of the termites Procubitermesaburiensis and Cubitermes severus

Abstract Isolates of the genus Streptomyces were readily obtained from the intestines of two African species of soil-feeding termites by an aerobic explant technique using starch casein medium, and from their parent soil and mound materials by dilution plating. Discriminant analysis of the isolates, based on 44 representative characters, showed that the population derived directly from the termites was significantly different from that of the feed soil or the mound. The termite gut was considered to be a good source of unusual actinomycetes, but strains isolated under aerobic conditions are likely to be allochthons selected by the intestinal environment, which is highly alkaline and anaerobic. An anaerobic, filamentous isolate was obtained which may be a component of the prokaryotic symbiont population mediating termite digestion.     

Nitrogen and carbon isotope ratios in termites: an indicator of trophic habit along the gradient from wood-feeding to soil-feeding

1. Nitrogen and carbon stable-isotope ratios (δ¹⁵N and δ¹³C) of body tissues, mound/nest materials and dietary substrates were determined in termite species with differing trophic habits, sampled from the Mbalmayo Forest Reserve, southern Cameroon.
2. δ¹⁵N of termite tissues was enriched gradually along a spectrum of species representing a trophic gradient from wood- to soil-feeding. Species that could be identified from their general biology and from gut content analysis as feeding on well-rotted wood or as wood/soil interface feeders showed δ¹⁵N intermediate between sound-wood-feeders and soil-feeders. It is proposed that δ¹⁵N is therefore a possible indicator of the functional position of species in the humification process. Differences in δ¹³C were also observed between wood-feeding and soil-feeding forms.
3. High values of δ¹⁵N in soil-feeding termites suggest that nitrogen fixation is of little importance in these species.
4. A wide range of isotope effects (the difference in isotope ratios between termites and their diet) was observed for both nitrogen (Δδ¹⁵N = –1.6 to + 8.8‰) and carbon (Δδ¹³C = –2.2 to + 3.0‰), which suggests a diversity of nutrient acquisition mechanisms within termites and diverse relationships between termites and their intestinal micro-organisms.     

Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus

Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of the Methanosarcinaceae, whereas Methanobacteriaceae and Methanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance of Thermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.     

On the elevated intestinal pH of higher termites (Isoptera: Termitidae)

Summary The pH of the gut contents was measured in 52 species of higher termites (Termitidae), representing 36 genera in all four subfamilies. A statistically significant trend was shown from lower termites with low mean gut pH through to the Termitinae with higher mean gut pHs. Elevation of the pH occurred principally in the first and third proctodaeal segments, reaching values as high as 10.5 in 8 soil-feeding genera and 1 wood-feeding genus of Termitinae. Elevation of gut pH within the Termitidae appears to be independent of the general nature of the feeding substrate.     

In vivo emission of dinitrogen by earthworms via denitrifying bacteria in the gut

Earthworms emit the greenhouse gas nitrous oxide (N2O), and ingested denitrifiers in the gut appear to be the main source of this N2O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N2), the end product of complete denitrification. When [15N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N2 (and also labeled N2O) under in vivo conditions; emission of N2 by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N2 per h per g (fresh weight), respectively. Isolated gut contents also produced [15N]nitrate-derived N2 and N2O under anoxic conditions. N2 is formed by N2O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [15N]nitrate-derived N2 by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N2O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N2 (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N2 per h per g (fresh weight), respectively; these emission rates approximated that of N2O. These collective observations indicate that (i) earthworms emit N2 concomitant with the emission of N2O via the in situ activity of denitrifying bacteria in the gut and (ii) N2O is quantitatively an important denitrification-derived end product under in situ conditions.     

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.     

Axial Differences in Community Structure of Crenarchaeota and Euryarchaeota in the Highly Compartmentalized Gut of the Soil-Feeding Termite Cubitermes orthognathus

Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of the Methanosarcinaceae, whereas Methanobacteriaceae and Methanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance of Thermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.