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.     

Nitrate reduction, nitrous oxide formation, and anaerobic ammonia oxidation to nitrite in the gut of soil-feeding termites (Cubitermes and Ophiotermes spp.)

Soil-feeding termites play important roles in the dynamics of carbon and nitrogen in tropical soils. Through the mineralization of nitrogenous humus components, their intestinal tracts accumulate enormous amounts of ammonia, and nitrate and nitrite concentrations are several orders of magnitude above those in the ingested soil. Here, we studied the metabolism of nitrate in the different gut compartments of two Cubitermes and one Ophiotermes species using (15)N isotope tracer analysis. Living termites emitted N(2) at rates ranging from 3.8 to 6.8 nmol h(-1) (g fresh wt.)(-1). However, in homogenates of individual gut sections, denitrification was restricted to the posterior hindgut, whereas nitrate ammonification occurred in all gut compartments and was the prevailing process in the anterior gut. Potential rates of nitrate ammonification for the entire intestinal tract were tenfold higher than those of denitrification, implying that ammonification is the major sink for ingested nitrate in the intestinal tract of soil-feeding termites. Because nitrate is efficiently reduced already in the anterior gut, reductive processes in the posterior gut compartments must be fuelled by an endogenous source of oxidized nitrogen species. Quite unexpectedly, we observed an anaerobic oxidation of (15)N-labelled ammonia to nitrite, especially in the P4 section, which is presumably driven by ferric iron; nitrification and anammox activities were not detected. Two of the termite species also emitted substantial amounts of N(2) O, ranging from 0.4 to 3.9 nmol h(-1) (g fresh wt.)(-1), providing direct evidence that soil-feeding termites are a hitherto unrecognized source of this greenhouse gas in tropical soils.     

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.     

Hydrogen Profiles and Localization of Methanogenic Activities in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

It has been shown that the coexistence of methanogenesis and reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes is based largely on the radial distribution of the respective microbial populations and relatively high hydrogen partial pressures in the gut lumen. Using Clark-type microelectrodes, we showed that the situation in Cubitermes orthognathus and other soil-feeding members of the subfamily Termitinae is different and much more complex. All major compartments of agarose-embedded hindguts were anoxic at the gut center, and high H₂ partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H₂. Posterior to the P3 segment, however, H₂ concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H₂ was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H₂ or formate was added. Addition of H₂ to the gas headspace stimulated CH₄ emission of living termites, indicating that endogenous H₂ production limits methanogenesis also in vivo. At the low H₂ partial pressures in the posterior hindgut, methanogens would most likely outcompete homoacetogens for this electron donor. This might explain the apparent predominance of methanogenesis over reductive acetogenesis in the hindgut of soil-feeding termites, although the presence of homoacetogens in the anterior, highly alkaline region cannot yet be excluded. In addition, the direct contact of anterior and posterior hindgut compartments in situ permits a cross-epithelial transfer of H₂ or formate, which would not only fuel methanogenesis in these compartments, but would also create favorable microniches for reductive acetogenesis. In situ rates and spatial distribution of H₂-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497–4505, 1999).     

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.     

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.     

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.     

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.     

Nitrogen cycling in forest soils across climate gradients in Eastern China

A ¹⁵N tracing study was carried out to investigate the potential gross nitrogen (N) dynamics in thirteen forest soils in Eastern China ranging from temperate to tropical zones (five coniferous forests, six deciduous broad-leaf forests, one temperate mixed forest, one evergreen broad-leaf forests ecosystems), and to identify the major controlling factors on N cycling in these forest ecosystems. The soil pH ranged from 4.3 to 7.9 and soil organic carbon (SOC) ranged from 6.6 g?kg^?1 to 83.0 g?kg^?1. The potential gross N transformation rates were quantified by ¹⁵N tracing studies where either the ammonium or nitrate pools were ¹⁵N labeled in parallel treatments. Gross mineralization rates ranged from 0.915 μg N g^?1 soil day^?1 to 2.718 μg N g^?1 soil day^?1 in the studied forest soils. The average contribution of labile organic-N (M _Nlab ) to total gross mineralization (M _Nrec +M _Nlab ) was 86% (58% to 99%), indicating that turnover of labile organic N plays a dominant role in the studied forest ecosystems. The gross mineralization rates in coniferous forest soils were significantly lower (ranging between 0.915 and 1.228 μg N g^?1 soil day^?1) compared to broad-leaf forest soils (ranging from 1.621 to 2.718 μg N g^?1 soil day^?1) (p?<?0.01). Thus, the dominant vegetation may play an important role in regulating soil N mineralization. Nitrate production (nitrification) occurred via two pathways, oxidation of NH ₄ ⁺ and organic N the forest soils. Correlations with soil pH indicated that this is a key factor controlling the oxidation of NH ₄ ⁺ and organic N in theses forest ecosystems. NH ₄ ⁺ oxidation decreased with a decline in pH while organic N oxidation increased. The climatic conditions (e.g. moisture status) at the various sites governed the NO ₃ ^? -N consumption processes (dissimilatory NO ₃ ^? reduction to NH ₄ ⁺ (DNRA) or immobilization of NO ₃ ^? ). Total NO ₃ ^? consumption and the proportion of total NO ₃ ^? consumption to total NO ₃ ^? production decreased with an increase in the drought index of ecosystems, showing that strong interactions appear to exist between climatic condition (e.g. the drought index), N mineralization and the rate of DNRA. Interactions between vegetation, climatic conditions govern internal N cycling in these forests soils.     

Methane Oxidation in Termite Hindguts: Absence of Evidence and Evidence of Absence

A steep oxygen gradient and the presence of methane render the hindgut internal periphery of termites a potential habitat for aerobic methane-oxidizing bacteria. However, methane emissions of various termites increased, if at all, only slightly when termites were exposed to an anoxic (nitrogen) atmosphere, and ¹⁴CH₄ added to the air headspace over live termites was not converted to ¹⁴CO₂. Evidence for the absence of methane oxidation in living termites was corroborated by the failure to detect pmoA, the marker gene for particulate methane monooxygenase, in hindgut DNA extracts of all termites investigated. This adds robustness to our concept of the degradation network in the termite hindgut and eliminates the gut itself as a potential sink of this important greenhouse gas.     

Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites

The redox potential and pH of the gut of the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding the insects with redox dyes and pH indicators. For N. exitiosus the E′₀ (pH 7.0) of the foregut was above +200 mV; the midgut was about +100 to +150 mV and the hindgut was in the region of ?20 to +30 mV. For C. lacteus the fore- and midgut were about +30 to +50 mV and the hindgut about ?20 to +20 mV. The colours of the ingested dyes indicated that the gut was aerobic in both termites. The pH of the whole gut ranged from 6.5 to 7.5 for N. exitiosus and 6.0 to 7.0 for C. lacteus.     

Nitrous Oxide (N2O) Emissions by Termites: Does the Feeding Guild Matter?

In the tropics, termites are major players in the mineralization of organic matter leading to the production of greenhouse gases including nitrous oxide (N₂O). Termites have a wide trophic diversity and their N-metabolism depends on the feeding guild. This study assessed the extent to which N₂O emission levels were determined by termite feeding guild and tested the hypothesis that termite species feeding on a diet rich in N emit higher levels of N₂O than those feeding on a diet low in N. An in-vitro incubation approach was used to determine the levels of N₂O production in 14 termite species belonging to different feeding guilds, collected from a wide range of biomes. Fungus-growing and soil-feeding termites emit N₂O. The N₂O production levels varied considerably, ranging from 13.14 to 117.62 ng N₂O-N d^-1 (g dry wt.)^-1 for soil-feeding species, with Cubitermes spp. having the highest production levels, and from 39.61 to 65.61 ng N₂O-N d^-1 (g dry wt.)^-1 for fungus-growing species. Wood-feeding termites were net N₂O consumers rather than N₂O producers with a consumption ranging from 16.09 to 45.22 ng N₂O-N d^-1 (g dry wt.)^-1. Incubating live termites together with their mound increased the levels of N₂O production by between 6 and 13 fold for soil-feeders, with the highest increase in Capritermes capricornis, and between 14 and 34 fold for fungus-growers, with the highest increase in Macrotermes muelleri. Ammonia-oxidizing (amoA-AOB and amoA-AOA) and denitrifying (nirK, nirS, nosZ) gene markers were detected in the guts of all termite species studied. No correlation was found between the abundance of these marker genes and the levels of N₂O production from different feeding guilds. Overall, these results support the hypothesis that N₂O production rates were higher in termites feeding on substrates with higher N content, such as soil and fungi, compared to those feeding on N-poor wood.     

Temperature sensitivity of soil carbon and nitrogen mineralization: impacts of nitrogen species and land use type

Background and aims

Climate warming, nitrogen (N) deposition and land use change are some of the drivers affecting ecosystem processes such as soil carbon (C) and N dynamics, yet the interactive effects of those drivers on ecosystem processes are poorly understood. This study aimed to understand mechanisms of interactive effects of temperature, form of N deposition and land use type on soil C and N mineralization.

Methods

We studied, in a laboratory incubation experiment, the effects of temperature (15 vs. 25 °C) and species of N deposition (NH₄ ⁺-N vs. NO₃ ^?-N) on soil CO₂ efflux, dissolved organic C (DOC) and N (DON), NH₄ ⁺-N, and NO₃ ^?-N concentrations using intact soil columns collected from adjacent forest and grassland ecosystems in north-central Alberta.

Results

Temperature and land use type interacted to affect soil CO₂ efflux, concentrations of DON, NH₄ ⁺-N and NO₃ ^?-N in most measurement times, with the higher incubation temperature resulted in the higher CO₂ efflux and NH₄ ⁺-N concentrations in forest soils and higher DON and NO₃ ^?-N concentrations in grassland soils. Temperature and land use type affected the cumulative soil CO₂ efflux, and DOC, DON, NH₄ ⁺-N and NO₃ ^?-N concentrations. The form of N added or its interaction with the other two factors did not affect any of the C and N cycling parameters.

Conclusions

Temperature and land use type were dominant factors affecting soil C loss, with the soil C in grassland soils more stable and resistant to temperature changes. The lack of short-term effects of the deposition of different N species on soil C and N mineralization suggest that maybe there was a threshold for the N effect to kick in and long-term experiments should be conducted to further elucidate the species of N deposition effects on soil C and N cycling in the studied systems.     

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.     

Novel lineages of Planctomycetes densely colonize the alkaline gut of soil-feeding termites (Cubitermes spp.)

Members of the phylum Planctomycetes are found in aquatic and terrestrial habitats. Here we show that the highest density of Planctomycetes in natural environments (2.6 × 10⁹ cells ml⁻¹) is encountered in the hindgut of soil-feeding termites ( Cubitermes spp.), where they constitute up to one-third of the bacteria in the alkaline P3 compartment detected by fluorescent in situ hybridization (FISH). A 16S-rRNA-based approach revealed that the planctomycete community is very diverse and falls into three major clusters representing novel, deeply branching lineages. Terminal restriction fragment length polymorphism (T-RFLP) analysis and FISH with cluster-specific oligonucleotide probes confirmed that most of the lineages are also present in other gut compartments, albeit in much lower numbers, but absent from the food soil. The majority of planctomycetes in the gut belong to a large clade, the 'Termite planctomycete cluster', which consists exclusively of clones from termite guts and seems to be represented in all termite species.     

Direct potentiometric determination of redox potentials of the gut contents in the termites Zootermopsis nevadensis and Cubitermes severus and in three other arthropods

A Pt and calomel electrode combination were used to determine the redox potentials of the gut contents in two termites, Zootermopsis nevadensis and Cubitermes severus. Strongly reducing conditions occurred in the paunch of Z. nevadensis (mean E_h = ?160 mV), consistent with many evidences that anaerobic fermentation of wood polymers occurs at this site. In C. severus, a soil-feeder, equivalent regions of the hindgut were more midly reducing (P1: mean E_h = ?104 mV; P3: mean E_h = ?47 mV) while the colon appeared microaerobic or aerobic. It is argued that these conditions are more appropriate to the digestion of humic materials. Potentials consistent with aerobic conditions were found throughout the guts of Periplaneta americana, Locusta migratoria and Glomeris marginata, although the cockroach hindgut was more reducing than the equivalent structures in the other non-termite species.     

Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H₂ or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H₂ was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H₂ sinks when external H₂ was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H¹⁴CO₃⁻. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H₂ was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H₂ was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H₂ or by a cross-epithelial H₂ transfer from the anterior gut regions, which may create microniches favorable for H₂-dependent acetogenesis.     

Determination of pH and oxygen status in the guts of lower and higher termites

The pH of the gut was determined in vitro in six species of termite by means of indicator dyes and a pH electrode. In the lower termite Zootermopsis nevadensis the pH was close to neutrality throughout, ranging 6.0–7.5, but in Reticulitermes lucifugus, acid conditions (pH 5.5–6.0) occurred in the crop and paunch. In the higher termites Nasutitermes costalis, Microcerotermes arboreus, Cubitermes severus and Procubitermes aburiensis, there was a common trend of incresing pH from the crop, which was slightly or moderately acidic, to the first proctodaeal segment (P1) where moderately (N. costalis) and strongly (M. arboreus, C. severus and P. aburiensis) alkaline conditions prevailed. A pH of 10.4 was measured in C. severus, equalling the highest recorded in any insect. In the posterior regions of the hindgut there was a return towards neutral or acidic conditions. When termite guts were homogenized with air-saturated Ringer's solution, the dissolved O₂ content of the Ringer's was reduced. This was shown to be largely attributable to an oxygen deficit generated within the gut in situ. The combined effects of strongly alkaline conditions and reduced oxygen tension on digestive processes and intestinal micro-organisms are discussed.     

Determination ofin situ KN by the chloroform fumigation-incubation method and mineralization of biomass N under anaerobic conditions

A silt loam soil from Pakistan was incubated at 30°C with increasing levels (67, 133, 200, 267 and 333 μg N g^?1 soil) of¹⁵N-labelled (NH₄)₂SO₄ and glucose (C/N ratio of 30 for all additions). At a stage when all of the applied¹⁵N was immobilized (transformed into microbial biomass), moist soil samples were subjected to the chloroform fumigation-incubation method (CFIM) for determination of K_N and microbial biomass. Mineralization of biomass derived from the applied¹⁵N and the native soil N was studied under anaerobic conditions. In situ values of K_N varied from 0.19 to 0.42 and increased with increasing levels of amendment (N + glucose). From 10 to 18% of the native soil N was found as microbial biomass. Anaerobic incubation of the soils resulted in the mineralization (determined as NH ₄ ⁺ ) of 15.08 to 29.23% of the biomass¹⁵N at different levels of amendment; 2.90 to 4.43% of the native soil N was mineralized. From 70 to 90% of the N mineralized from native soil N was derived from microbial biomass; the rest was attributed to non-biomass N.