in the highly compartmentalized gut of Cubitermes spp.
Dissertation
zur Erlangung des Grades eines Doktors der Naturwissenschaften im Fachbereich Biologie der naturwissenschaftlichen Sektion
der Universität Konstanz
vorgelegt von Dirk Schmitt-Wagner
Tag der mündlichen Prüfung: 17. Juni 2003 Referent: Priv.-Doz. Dr. Andreas Brune
Referent: Prof. Dr. Bernhard Schink
Konstanz 2003
Contents
1
Introduction _________________________________________________________________________ 32
Hydrogen profiles and localization of methanogenic activities in the highly compartmentalized hindgut of soil-feeding highertermites (Cubitermes spp.) ________________________________________________11
3
Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus____________________________________ 29
4
Phylogenetic diversity, abundance, and axial distribution of microorganisms in the intestinal tract of soil-feeding termites(Cubitermes spp.)__________________________________________________________________ 53
5
Axial dynamics, stability, and inter-species similarity of bacterial community structure in the highly compartmentalized gut ofsoil-feeding termites (Cubitermes spp.) ______________________________________ 77
6
Axial distribution and phylogenetic diversity of spirochetes inthe hindgut of the soil-feeding termite Cubitermes ugandensis__________ 91
7
Discussion _______________________________________________________________________107Summary ________________________________________________________________________________ 119 Zusammenfassung_____________________________________________________________________ 121 Curriculum vitae _______________________________________________________________________ 123 Abgrenzung der Eigenleistung ______________________________________________________ 124
Significance of termites in the ecosystem
Termites, which belong to the order Isoptera, are terrestrial, social insects, phylogenetically related to cockroaches and mantids. Termites comprise 281 genera and over 2600 described species. At present, seven families and 14 subfamilies are recognized, but a large majority (ca. 85%) of genera described to date are included in only one family, the Termitidae. (Krishna, 1970; Wood and Johnson, 1986; Noirot, 1992; Abe et al., 2000; Kambhampati and Eggleton, 2000; see Fig. 1).
Fig. 1. Phylogenetic scheme of termite evolution showing the presumed relationship of the seven termite families, adapted from Higashi and Abe (1997). The numbers on the lines represent the number of genera/species in the different families (Abe et al., 2000).
The majority of termites live in tropical and subtropical regions, but they spread also into the temperate zone. About two-thirds of the Earth's land surface between the latitudes 48° N and 45° S is inhabited by termites (Lee and Wood, 1971). In tropical and subtropical regions, their number exceeds 6000 individuals m–2, and their biomass densities range between 5 and 50 g m–2, often surpassing biomass densities of mammalian herbivores (0.01–17.5 g m–2; Lee and Wood, 1971; Collins, 1989).
Termites can be classified in phylogenetically lower and higher termites. Lower termites comprise six families and are restricted to a diet of wood or grass (Noirot 1992), whereas higher termites (Termitidae) include soil-feeding (humivorous), wood- and grass-feeding (xylophagous) and fungus-cultivating species (Noirot 1992).
It is widely accepted that termites have a major impact on the decomposition of plant material, humification, and soil conditioning (Lee and Wood, 1971; Wood and Sands, 1978; Wood, 1988; Collins, 1989; Martius, 1994; Brussaard and Juma, 1996).
Lower termites Cockroaches
Isoptera (termites)
Mastotermitidae Kalotermitidae Hodotermitidae Termopsidae Rhinotermitidae
Termitidae Serritermitidae 1/1
21/411
3/15
5/20 16/305
1/1 236/1895
Higher termites
Therefore, termites take actively part in the permanent alteration of their habitat, especially in tropical ecosystems.
Approximately 136 × 1015 g of carbon dioxide is fixed annually by photosynthesis in form of plant biomass. The major constituents of plant biomass are cellulose and lignin. About two-thirds of this biomass production occurs in terrestrial ecosystems (Breznak and Brune, 1994 and references therein). The decomposition of plant material is carried out primarily by fungi and bacteria, but termites, as the most abundant and important soil macro-invertebrates, also play an important role in this process. Like shredder organisms, termites can mechanically chop up the plant material with their mandibles and their gizzard, thereby increasing the surface area accessible to soil microorganisms. Furthermore, termites can also directly dissimilate the structural polymers of lignocellulose with the help of their gut symbionts (Lee and Wood, 1971; Breznak and Brune, 1994).
Due to the high biomass densities, termites significantly contribute to the emission of atmospheric trace gases like methane and carbon dioxide. The first studies had estimated the contribution of termites to the global methane emission up to 45% (Zimmerman et al., 1982), but more recent estimations based on a larger data set, and the consideration of severe differences in methane emission rates between different termite species, reduced their contribution to less than 5% (Sanderson, 1996; Bignell et al., 1997; Sugimoto et al., 1998).
Soil feeding termites
Humivorous termites represent one of the most abundant and ecologically important groups of soil macroinvertebrates in tropical ecosystems. In contrast to the phylogenetically lower termites, which are restricted to a diet of wood, the majority of species of the higher termites (family Termitidae) consume soil organic matter in various stages of humification. True soil feeders occur in all subfamilies accept the Macrotermitinae (Abe et al., 2000).
The humivorous mode of nutrition rendered the Termitidae independent of the necessity to harbor cellulolytic flagellates as symbionts, and thereby probably removed important evolutionary constraints, allowing further diversification of the gut (Noirot, 1992). Especially in the true soil feeders, the increase in length, volume, and compartmentalization transformed the hindgut into a complex microecosystem with pronounced axial dynamics of the intestinal pH, ranging from slightly acidic conditions in the crop and the P5 segment to extremely alkaline conditions in the P1 and the P3 segment (Bignell and Eggleton, 1995; Brune and Kühl, 1996; see Fig. 3.).
Soil-feeding termites ingest large amounts of soil (Wood, 1978a; Wood, 1978b;
Okwakol, 1980), and due to their high biomass densities, their feeding activity is important for the biomass turnover in tropical and subtropical ecosystems (Wood and Johnson, 1986; Wood, 1988; Collins, 1989; Martius, 1994; Bignell et al., 1997;
Abe et al., 2000). The food ingested by soil-feeding termites is quite heterogeneous.
Fig. 2. Phylogenetic scheme of the presumed relationship of the four subfamilies of the higher termites (Termitidae), adapted from Bignell and Eggleton (1995). The number on the lines represent the number of genera/species in the respective branch (Abe et al., 2000). The subfamily Termitinae includes the species of the genus Cubitermes which were the objects of this study.
The gut contains predominantly soil minerals and humus, but also plant tissue fragments, plant roots, fungal mycelia, and macerated organic material (Sleaford et al., 1996; Donovan et al., 2001). However, the identity of the components used as electron and carbon sources was obscure for a long time (Bignell et al., 1997). Some authors assumed that the aromatic components of the humic substance fraction could contribute to feed the humivorous termites (Bignell, 1994). In a recent study it was shown, that the extreme alkalinity in the anterior hindgut facilitates not only the desorption of humic substances from the mineral matrix, but also decreases their molecular weight and increases their solubility (Kappler and Brune, 1999).
Fig. 3. Gut morphology of a Cubitermes sp. worker termite, representative also for other soil- feeding Termitinae. The gut was drawn in its unraveled state to illustrate the various segments: C (crop), M (midgut), ms (mixed segment), P1–5 (proctodeal segments). The average luminal pH was determined for the indicated gut regions in Cubitermes speciosus using intact guts and glass pH microelectrodes (Brune and Kühl, 1996).
In feeding experiments with chemically identical synthetic humic acids, radioactively labeled in their proteinaceous or aromatic building blocks, the mineralization rate of the peptidic components of the humic substances increased dramatically in the presence of termites, whereas the mineralization rate of the aromatic components increased only weakly (Ji et al., 2000). The results of this study
Higher termites Macrotermitinae
Apicotermitinae
Nasutitermitinae
Termitinae Termitidae
15/332
43/196
90/659
88/708 236/1895
were a first indication of the importance of peptidic components of humic acids as substrates for soil-feeding termites.
In soil inoculated with radioactively labeled preparations of cellulose, peptidoglycan, protein and whole bacterial cells, the mineralization rate of all these compounds was strongly increased when termites were present. Consequently, also structural polysaccharides of plant and microorganisms and microbial biomass could be a nutrition source of importance for soil-feeding termites (Ji and Brune, 2001).
Soil-feeding termites emit more methane than wood-feeding termites, indicating that methanogenesis represents the major hydrogen sink reaction in the gut, although only few termite species were tested so far (Brauman et al., 1992). A study using microinjection into intact guts showed, that methanogens outcompete homoacetogens for the endogenous reductant in the hindgut of soil-feeding termites also under in situ conditions (Tholen and Brune, 1999).
The aims of these studies
In contrast to lower termites (Ohkuma et al., 1995; Ohkuma and Kudo, 1996; Kudo et al., 1998; Ohkuma and Kudo, 1998; Shinzato et al., 1999; Shinzato et al., 2001;
Hongoh et al., 2003), diversity and community structure of the microbiota in the highly compartmentalized gut of higher soil-feeding termites is largely unknown and also the role of the gut microflora in the digestion process is still obscure (Bignell et al., 1997), although it was reported that the intestinal tract of soil-feeding termites contains a high density of microbial cells (Bignell et al., 1980), and also the high concentrations of microbial fermentation products in the individual gut compartments indicate the presence of an active gut microbiota (Tholen, 1999; E.
Miambi, A. Tholen, H. Boga, and A. Brune; unpublished results).
In view of the specific morphological and physicochemical adaptations of the digestive system, the necessity of a specific gut microbiota in soil-feeding termites has been questioned (Bignell, 1994), and it cannot be excluded that the microorganisms in the gut represent a transient microbiota of soil microorganisms proliferating under favorable conditions in certain compartments of the intestinal tract.
The aims of the studies summarized in this thesis were to describe the diversity of the gut microbial community, to localize specific microbial processes such as hydrogen production and methanogenesis, and to determine the physicochemical conditions with specific microsensors, focusing on the description of the distribution of hydrogen sources and potential hydrogen sinks among the different gut compartments.
Since cultivation-based studies covered only a small proportion of the gut microorganisms present, the archaeal and bacterial diversity and the absolute abundance and axial distribution of the major phylogenetic groups had to be determined with molecular biology techniques, such as clonal analysis of archaeal and bacterial 16S rRNA genes, fluorescence in situ hybridization (FISH) of microbial
cells, and terminal restriction fragment length polymorphism (T-RFLP) fingerprints of archaeal and bacterial 16S rRNA genes in the major gut compartments of soil- feeding termites.
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activities in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.)
Dirk Schmitt-Wagner and Andreas Brune
Published in Applied and Environmental Microbiology 65, 4490–4496 (1999)
Abstract
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).
Introduction
In the metabolic reactions involved in lignocellulose degradation in termite hindguts, hydrogen appears to be the key intermediate linking the fermentative breakdown of carbohydrates with methanogenesis and reductive acetogenesis. In lower termites, H2 formation is mainly attributed to the dense populations of cellulolytic flagellates which are characteristic for this group. In higher termites, symbiotic flagellates are absent, and the reactions responsible for the formation of H2 are not known.
Reductive acetogenesis and methanogenesis, however, occur in all termites investigated to date, and are considered typical for the strictly anaerobic metabolic activities in termite guts (for reviews, see references Breznak, 1994; Breznak and Brune, 1994; Brune, 1998).
In earlier studies, it was generally assumed that the H2 partial pressure in termite guts is very low. Since methanogens would be expected to outcompete homoacetogens for H2 in a homogeneous well-mixed system simply for thermodynamic reasons (Schink, 1997), it was enigmatic why reductive acetogenesis apparently predominates over methanogenesis as the major hydrogen sink reaction in the guts of many wood-feeding termites (Brauman et al., 1992; Breznak, 1994).
However, recent studies with microsensors have revealed that termite hindguts are by far not homogeneous anoxic fermentors, but are highly structured environments characterized by steep gradients of O2, H2, and pH (Brune et al., 1995; Brune and Kühl, 1996; Ebert and Brune, 1997). In the case of the wood-feeding lower termite Reticulitermes flavipes, the hindgut paunch exhibits luminal H2 partial pressures as high as 5 kPa (Ebert and Brune, 1997). The methanogenic microbial population of this termite is located almost exclusively within the microoxic periphery of the hindgut, i.e., directly at the gut epithelium (Leadbetter and Breznak, 1996), where it represents a major hydrogen sink and gives rise to steep H2 gradients towards the gut wall (Ebert and Brune, 1997). The homoacetogenic microorganisms, however, are most likely located within the gut lumen, where they would benefit from the high H2 partial pressure (Ebert and Brune, 1997; Leadbetter et al., 1999). On the basis of these results, it was postulated that the coexistence of homoacetogens and methanogens within the hindgut of this termite is not based on direct competition for a limited substrate, but rather on resource partitioning effected by the spatial distribution of the different H2-consuming populations within the gut (Ebert and Brune, 1997; Brune, 1998). It is not clear whether the situation in R. flavipes can be generalized for other wood-feeding termites.
In contrast to lower termites, the majority of the most abundant and ecologically important family of higher termites (Termitidae) are soil-feeding (Wood and Johnson, 1986). A humivorous mode of nutrition is attributed to the majority of all genera in three of the four subfamilies, Apicotermitinae (95%), Termitinae (74%), and Nasutitermitinae (44%), whereas no soil feeders are found among the fungus- cultivating Macrotermitinae (Noirot, 1992). The hindguts of soil-feeding termites are
highly compartmentalized and characterized by an unusually high pH in their anterior region (Bignell and Anderson, 1980; Bignell, 1994; Bignell and Eggleton, 1995; see also Fig. 1). In soil-feeding Termitinae, the luminal pH increases sharply in the mixed segment, a gut region located between the neutral midgut and the first proctodeal dilation (P1), which possesses the highest alkalinity ever observed in biological systems (pH >12; ref. Brune and Kühl, 1996).
Soil-feeding termites generally show higher methane emission rates and lower potential rates of H2-dependent acetogenesis than wood-feeding species, indicating that in this feeding guild methanogenesis represents the major hydrogen sink reaction in the hindgut (Brauman et al., 1992; Rouland et al., 1993). In view of the enormous biomass of termites and their keystone role in decomposition processes in tropical ecosystems, it is not astonishing that their CH4 emissions are considered to contribute significantly to the global fluxes of this atmospherically relevant trace gas (for a review, see references Sanderson, 1996 and Bignell et al., 1997). It is therefore important to understand the physiological and autecological factors that regulate the coexistence of the H2-oxidizing populations in the gut microbial community of these termites. To date, it is completely unknown whether H2 accumulates to significant concentrations in any of the hindgut compartments, whether homoacetogens coexist with methanogens in the same compartment(s), or whether each group is localized in different gut regions. Since the existing studies have so far compared CH4 emission of living termites with H2-dependent acetogenesis in gut homogenates, it can also not be excluded that the importance of homoacetogens in soil-feeding termites has been underestimated.
In this study, we used Clark-type microelectrodes to characterize the axial and radial profiles of O2 and H2 partial pressure in the gut of soil-feeding Cubitermes spp., focusing on the distribution of hydrogen sources and potential hydrogen sinks among the different gut compartments with respect to the localization of the methanogenic activities. In a parallel study described in a companion paper, we used a newly developed technique involving microinjection of radiotracers to determine in situ rates of reductive acetogenesis and the exact location of homoacetogens within the gut (Tholen and Brune, 1999).
Materials and Methods Termites
Cubitermes orthognathus EMERSON and Cubitermes umbratus WILLIAMS were collected near Busia (Kenya) and in the Shimba Hills Natural Reserve (Kenya), respectively;
Thoracotermes macrothorax (SJÖSTEDT), Noditermes indoensis SJÖSTEDT, and Procubitermes sp. (all Termitidae: Termitinae) were collected in the Mayombe rain forest (Congo- Brazzaville). Anoplotermes sp. (Termitidae: Apicotermitinae) was collected in a coastal rain forest in southern Brazil, and tentatively identified as A. pacificus FR. MÜLLER on the basis of the close association of the nest with tree roots (Kaiser, 1953). The
termites were brought to the laboratory in polypropylene containers together with nest fragments and soil of the original collection site; measurements were generally performed within 1–2 months of collection. Reticulitermes flavipes (KOLLAR) (Rhinotermitidae) and Nasutitermes arborum (SMEATHMAN) (Termitidae:
Nasutitermitinae) were from birch-fed laboratory cultures. Worker caste termites were used for all experiments.
Microelectrode measurements
Clark-type oxygen microelectrodes with guard cathodes (Revsbech, 1989) were constructed in our laboratory and calibrated as described (Brune et al., 1995).
Hydrogen microelectrodes had the same principal design (Witty, 1991), except that the working electrode was platinum-coated as described by Ebert and Brune (Ebert and Brune, 1997). Testing and calibration procedures were as previously described (Ebert and Brune, 1997). Both types of microelectrodes had tip diameters of 10–
20 µm.
The experimental setup for the measurements was essentially as described previously (Brune et al., 1995). The bottom of a glass-faced microchamber (Ebert and Brune, 1997) was filled 2–4 mm deep with a layer of agarose (1%) made up with modified insect Ringer's solution (Brune et al., 1995). A freshly dissected termite gut was placed flat and fully extended onto the agarose, and was quickly covered with an shallow layer (1–2 mm) of identical agarose (40°C), which cooled and solidified immediately. Both the microchamber and the agarose were electrically grounded for static discharge. Experiments with defined gas headspace were performed using a glass bell placed over the microchamber, which was continuously flushed with the desired gas mixture (setup described in detail in reference (Ebert and Brune, 1997).
Microelectrodes were positioned with a manual micromanipulator (MM33;
Märzhäuser, Wetzlar, Germany); all measurements were performed at room temperature (20–22°C).
Estimation of hydrogen fluxes
Gut segments were approximated as endless cylinders, and the H2 flux from or into each segment was estimated from the slope of the radial H2 concentration profiles directly above the gut, using Fick's first law of diffusion:
J = –2π r l φ Ds δC (r) / δr (Crank, 1975), where J is the total flux of molecules through the surface per unit of time, r and l are segment radius and length, φ is the porosity of the agarose, Ds is the apparent diffusion coefficient, and δC(r) / δr is the slope of the concentration gradient at radius r. The calculation details were exactly as previously described (Ebert and Brune, 1997).
Gas emission by termites and gut sections
Between 10 and 20 termites (60 for A. pacificus) were placed into small glass vials (10 ml). In the case of C. orthognathus, 20 guts were also dissected and separated into
five sections representing the major gut compartments (Fig. 1), which were carefully placed onto filter paper strips (Whatman No. 1) soaked with buffered salts solution (BSS; ref. Tholen et al., 1997). The paper strips were then placed into separate vials containing a shallow layer of BSS. The P3 and the P4a segments were not separated to avoid leaking of the gut contents. For the measurements under anoxic conditions, the complete preparation procedure was performed in an anoxic glove box with a N2 atmosphere. All vials were closed with rubber septa; when indicated, the gas headspace was exchanged by flushing with the desired gas mixture for 1 min. The vials were incubated at room temperature (22 ± 1 °C). For a period of 1.5 to 2 h, headspace samples (300 µl) were taken at regular intervals and immediately replaced with an equal volume of the original headspace mixture, using a syringe equipped with a gas-tight valve. CH4 and H2 concentrations were determined by gas chromatography on a molecular sieve column using flame-ionization detection (Platen and Schink, 1987) and a HgO-reduction trace gas analyzer (Friedrich and Schink, 1993), respectively; the gas emission rates were corrected for the dilution effects caused by the sampling procedure. All gases were supplied by SWF, Friedrichshafen, Germany, and were 99.999% pure.
Fig. 1. Gut morphology of a Cubitermes sp. worker termite, representative also for other soil- feeding Termitinae. The gut was drawn in its unraveled state to illustrate the various segments: C (crop), M (midgut), ms (mixed segment), P1–5 (proctodeal segments). The average luminal pH was determined for the indicated gut regions in Cubitermes speciosus using intact guts and glass pH microelectrodes (Brune and Kühl, 1996). When gut sections were used, the guts were separated at the indicated positions.
Results
Axial profiles
Freshly prepared guts of soil-feeding Termitinae embedded in agarose–Ringer's solution remained physiologically active for more than 1 h, as indicated by the stable oxygen and hydrogen gradients between hindgut and agarose surface, which usually reached a steady state within 5–10 min, and by (although infrequent) hindgut peristalsis, mainly in the posterior segments. In this respect, the results were similar to those previously obtained with wood-feeding termites (Brune et al., 1995; Ebert and Brune, 1997). Only the results with Cubitermes orthognathus are presented in detail,
Fig. 2. Axial profiles of oxygen (A) and hydrogen (B) partial pressure in agarose-embedded guts of Cubitermes orthognathus. H2 measurements were performed under air (z) or under N2 headspace ({). The graphs represent typical profiles obtained with freshly fed termites. All values are for the gut center. The borders between gut segments are indicated by vertical dotted lines; see Fig. 1 for abbreviations.
although axial and radial profiles of Cubitermes umbratus and Thoracotermes macrothorax guts gave essentially the same results.
Axial oxygen profiles revealed that all gut compartments except the tubular midgut and the less-dilated regions of the posterior hindgut (Fig. 1) were anoxic at the gut center (Fig. 2A). Hydrogen accumulated only in the anterior gut region, with the highest H2 partial pressures in the mixed segment and the P3 (Fig. 2B). In the P4a and P4b, H2 was always below the detection limit (100 Pa). The low values obtained in the rectum (P5) may have to be regarded with caution since the termites frequently voided their rectal contents during dissection. When the measurements were performed under anoxic conditions, the H2 partial pressures in the anterior gut regions increased significantly; only in the P3 was this effect less pronounced.We found that H2 partial pressures in the anterior hindgut decreased progressively with time already during the first weeks after collection; this phenomenon was most pronounced in case of the P3. However, high H2 partial pressures in the P3 of Cubitermes orthognathus, which ranged from 0.1–1 kPa in starved individuals, could be
0 2 4 6 8 10
O2 partial pressure (kPa)
P5 P4b
P4a P3 P1 ms M C
A
0 1 2 3 4 5
0 2 4 6 8 10 12 14 16 18
Relative distance (mm)
H2 partial pressure (kPa) B
restored by feeding topsoil from the collection site to small batches of termites.
Within 24 h, the H2 partial pressure in the P3 rose to maximum values (1.5–9 kPa), which were even higher than those measured two weeks after collection. Apparently, intestinal H2 production in soil-feeding Termitinae is severely affected by the starvation situation with which the termites are inevitably faced in the laboratory.
Unfortunately, all long-term feeding attempts by us, and to our knowledge also by others, have so far been unsuccessful.
Radial profiles
The negative slopes of the O2 profiles from the agarose surface toward the gut wall indicated that all hindgut regions were significant oxygen sinks (Fig. 3A). The gradients were quasi-linear at a distance, but, due to the radial symmetry of the system, increased in curvature close to the gut wall, which was most obvious when the gut diameter was small. The sharp changes in slope at the perimeter of the P1 and the P3, however, which were previously observed also with Cubitermes speciosus (Kappler and Brune, 1999), indicate a smaller diffusion coefficient in these gut regions, probably caused by a lower porosity of the tightly packed gut contents. In the large proctodeal compartments P1 and P3, O2 often penetrated significantly into the gut, especially in starved termites, rendering only the gut center completely anoxic. The highest hydrogen partial pressures were always found at the center of the mixed segment and the P3 segment (Fig. 3B). The steep concentration profiles indicated strong fluxes of H2 directed towards the gut periphery, which generally continued into the agarose. In the case of the mixed segment, the H2 efflux from the gut increased together with the luminal H2 partial pressure when the guts were incubated under anoxic conditions. Hydrogen accumulation and H2 fluxes from the gut proper into the agarose were less pronounced in the P1, and were never observed in the posterior compartments, P4a and P4b.
Uptake of exogenously supplied hydrogen
When exogenous H2 was provided via the headspace gas, all hindgut regions turned into hydrogen sinks, especially those which accumulated no or only small amounts of H2 from internal sources (Fig. 3C). Consumption of H2 was generally more pronounced when it was supplied in the presence of air than under nitrogen, and in the hindgut compartments posterior to the P3 external H2 was often completely consumed already in the gut periphery when O2 was present. Only in the case of the mixed segment incubated under anoxic conditions, endogenous H2 production caused H2 concentrations at the gut center despite the presence of exogenous H2; a comparison with the profile under air indicates a mainly aerobic nature of the H2- consuming activities in the mixed segment.
Hydrogen fluxes were estimated from the slopes of the gradients directly above each gut segment. Highest H2 emission rates were obtained with the P3, which was followed by the mixed segment, whereas those of midgut and P1 were much lower
Fig. 3. Radial profiles of the oxygen (A) and hydrogen partial pressure (B, C) around and within different hindgut segments of Cubitermes orthognathus. The guts were embedded in agarose; the shaded areas indicate the position of the gut proper in the surrounding agarose, represented by the white background. One axis tick represents a distance of 250 µm.
Oxygen profiles (A) were measured under an air headspace, hydrogen profiles either under air (z) or under N2 ({), without (B) or with the addition of H2 (20 kPa) to the headspace gas (C). In the case of the mixed segment (ms), also a N2 headspace with 5 kPa H2 was used ( ).
The graphs represent typical sets of similar profiles obtained in numerous experiments with different guts.
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(Fig. 4A). When incubated under anoxic conditions, the H2 emission rates of the anterior segments increased more than twofold, only those of the P3 were not significantly affected. When exogenous H2 was added, all major hindgut compartments consumed H2 with roughly similar rates when compared on a per segment basis (Fig. 4B). Under oxic conditions, the sum of potential H2 uptake rates of all hindgut compartments was more than an order of magnitude higher than the efflux rates of endogenously produced H2 from the anterior region.
Methane and hydrogen emission by living termites
The specific rates of CH4 emission of all termites species included in this study were roughly in the same range as their H2 emission rates; the emission rates for both gases increased only slightly when the termites were incubated under anoxic conditions (Table 1). The average of the methane emission rates of the soil-feeding species included in this study (0.180 ± 0.048 µmol g–1 h–1; air headspace) is only
Fig. 4. Hydrogen emission rates (A) and potential hydrogen uptake rates (B) of different gut regions of Cubitermes orthognathus, calculated from the slopes of the H2 gradients directly above each gut segment (see Fig. 3 for examples) and by approximating each segment as an endless cylinder. The gas headspace consisted of air (closed bars) or N2 (open bars); for H2 uptake rates, H2 (20 kPa) was added to the headspace gas. The values are means (± SD) of 3–5 different profiles.
slightly lower than that of the rates reported by Nunes et al. (1997) for a larger set of "field-sampled" species (0.238 ± 0.059 µmol g–1 h–1), whereas both data sets fall significantly behind the average of the methane emission rates found by Brauman et
0 1 2 3 4 5
M ms P1 P3 P4a P4b
H2 efflux rate (nmol h–1 ) A
0 2 4 6 8 10 12
M ms P1 P3 P4a P4b
H2 uptake rate (nmol h–1 ) B
Table 1. Hydrogen and methane emission rates of living termites under different headspace gases. H2 emission rate b CH4 emission rate b Termite species aFresh wt. (mg) Air N2n Air N2nAir + H2 c N2 + H2 cn Nasutitermes arborum (w) 2.8 122 r 22 180 r 16 4143 149 1 784 r 28 838 r 31 2 Reticulitermes flavipes (w) 4.0 165 r 68 244 r 128 8116r 22 131 r 8 4 587 r 180 695 r 133 4 Anoplotermes pacificus (s)1.1 195 r 64 399 r 146 5260r 69 369 r 72 2 1674 r 110 1831 r 266 2 Cubitermes orthognathus (s) 6.8 ND d ND 158 ± 28 160 ± 29 3 1123 ± 88 1176 ± 254 3 Noditermes sp. (s)3.7 285 r 110 449 r 219 3134 196 1 1338 r 352 1311 r 20 3 Procubitermes sp. (s)11.5 127 r 48 196 r 54 3181 200 1 1015 r 165 1462 r 169 3 Thoracotermes macrothorax (s)11.8 122 r 95 225 r 56 3168r 35 238 3 1461 r 338 1372 r 316 3 a Wood-feeding (w) or soil-feeding (s) habit is indicated in parentheses. b Rates are given in nmol (g fresh wt.)–1 h–1 and are means ± standard deviations of n independent assays. c H2 partial pressure was 20 kPa. d ND; not determined
Table 2. Methane emission rates of individual gut sections of Cubitermes orthognatus compared to those of living termites. a
CH4 emission rate (nmol termite–1 h–1) b Termite
type or
section Air N2 Air + H2 N2 + H2 N2 + formate Live 1.09 ± 0.11 1.12 ± 0.13 8.02 ± 0.09 8.09 ± 1.16 NA c
M – d – – – ND e
P1 0.09 ± 0.01 0.09 ± 0.01 0.11 ± 0.01 0.11 ± 0.02 0.15 P3/4a 0.21 ± 0.04 0.57 ± 0.13 1.51 ± 0.09 2.43 ± 0.72 4.73 P4b 0.02 ± 0.02 0.09 ± 0.02 0.13 ± 0.09 0.47 ± 0.11 0.51
P5 – – – 0.05 ± 0.01 ND
a Gut sections were incubated for 1.5 to 2 h and suspended in a shallow layer of BSS. The gas headspace consisted of air or N2, with or without the addition of H2 (20 kPa); formate (5 mM) was added to BSS.
b Balues are means ± mean deviations of two independent assays.
c NA, not applicable.
d –, below the detection limit (0.02 nmol termite-1 h-1).
e ND; not determined.
al. (1992) for a comparable set of species sampled "directly from the nest"
(0.730 ± 0.270 µmol g–1 h–1).
This may be related to our observation that both the CH4 and H2 emission rates of soil-feeding termites decreased progressively after the time of collection. Four months after collection, H2 emission rates of Thoracotermes macrothorax and Noditermes sp. had decreased to 13–14% of the values determined two weeks after collection.
Also the H2 partial pressures in the anterior hindgut were much lower than in the first weeks after collection, probably caused by an insufficient supply of fermentable substrates in laboratory colonies which are presumably feeding mainly on mound material (see above). The hydrogen limitation of methanogenesis was completely relieved when exogenous H2 was provided in the headspace gas. In all termite species tested, including well-nourished wood-feeding laboratory cultures taken directly from their substrate, CH4 emission rates increased 5- to 10-fold, and were almost unaffected by the presence of oxygen (Table 1).
Methane formation by gut sections
In order to localize the methanogenic activities within the gut, we determined the CH4 production rates for individual gut sections of C. orthognathus incubated under oxic or anoxic conditions and in the presence or in the absence of externally supplied H2 (Table 2). The highest CH4 emission rates of all sections were observed with the P3/4a compartment, whereas only low rates were observed with the P1 and the P4b
compartments. In case of P4b, however, high potential methanogenic activities became apparent when external H2 or formate was added. Also the CH4 emission rates of the P3/4a section was strongly stimulated by external H2 or formate.
Generally, H2-dependent methanogenesis was slightly higher in the absence of O2, and stimulation of methanogenesis by formate was higher than by H2.
Methane emission rates of living termites in the presence of external H2 were two- to threefold higher than the sum of the CH4 emission rates of separated gut segments under the same conditions, indicating that in the case of separated gut segments, the CH4 production rates are underestimated. This is not astonishing considering that the shallow but unstirred layer of buffered salt solution covering the gut sections represents a considerably larger diffusion barrier for hydrogen than the tracheal system of the termites, where virtually all diffusive transport occurs in the gas phase.
Discussion
This is the first report on hydrogen partial pressures in the hindgut of soil-feeding termites. Results of a previous study, which so far represented the only study of this subject in insect guts, had shown that H2 accumulates to high concentrations in the hindgut paunch of the lower termite Reticulitermes flavipes (Ebert and Brune, 1997) and had helped considerably in resolving the enigmatic predominance of reductive acetogens in wood-feeding termites (Brune, 1998; Tholen and Brune, 2000). Our current study demonstrates that the situation in the soil-feeding Termitinae is much more complex. In this group of termites, the hindgut is differentiated into several consecutive compartments, two of which are extremely alkaline (P1, P3). Despite the anoxic conditions at the center of all major hindgut dilations, only those anterior to the P4a segment accumulated H2 to significant partial pressures, whereas H2 was generally below the detection limit in all hindgut compartments posterior to the P3 segment.
The posterior gut sections (P3/4a and P4b) contained also virtually all the methanogenic activities in the hindgut. The efficient sinks for exogenous H2 in all hindgut compartments, the large CH4 emission rates of the isolated P3/4a section in the absence of exogenous electron donors, and the stimulation of CH4 emissionby externally added H2 are strong indications that the low H2 partial pressures in the P4a are not due to the absence of H2-forming activities but rather to the efficient removal of endogenously formed H2, analogous to the situation in anoxic sediments (Conrad, 1995). In contrast, endogenous sources of reducing equivalents in the P4b section seem to be negligible, since its methanogenic capacities became evident only when exogenous H2 or formate was added to the isolated gut sections.
Methane emission rates of all termite species tested in the present study increased considerably when living termites were incubated in the presence of exogenous H2, which is in agreement with two previous reports for Reticulitermes flavipes (Ebert and Brune, 1997) and Zootermopsis angusticollis (Messer and Lee, 1989). It appears that both
in lower and higher termites, irrespective of their feeding guild, methanogenic activities are strongly hydrogen-limited. Nevertheless, all termites tested also emitted H2 at rates which were in the same range as those of CH4 emission (Table 1).
Fig. 5. In situ orientation of the different gut segments within the abdomen of a Cubitermes sp. worker termite. Both dorsal (A) and ventral (B) aspects are shown; for segment nomenclature, see Fig. 1.
Hydrogen emission by the wood-feeding Reticulitermes flavipes has been attributed to the considerable accumulation of H2 in the lumen (Ebert and Brune, 1997) and a certain degree of patchiness in the epithelial colonization by methanogenic archaea (Leadbetter and Breznak, 1996), which represent a major hydrogen sink in the gut periphery (Ebert and Brune, 1997) and probably control H2 efflux. In soil-feeding Termitinae, however, there is an axial separation of H2-producing and H2-consuming activities, and the hydrogen profiles do not signify the presence of efficient H2 sinks at the epithelium of the anterior hindgut compartments (Fig. 3). This is in agreement with microscopic observations indicating the absence of any gut wall colonization in the P1 and P3 segments of Procubitermes aburiensis and Cubitermes umbratus (Bignell et al., 1980; Tholen and Brune, unpublished results) and with the large amounts of endogenous H2 escaping from isolated guts (Fig. 4). At first glance, it is therefore quite astonishing that the H2 emission rates of soil-feeding termites did not significantly exceed the values observed with the wood-feeding Reticulitermes flavipes (Table 1) or other wood- and litter-feeding species (Odelson and Breznak, 1983;
Williams et al., 1994; Anklin-Mühlemann et al., 1995; Nunes et al., 1997).
Most probably, the explanation lies in the proximity of the different hindgut regions within the abdomen of the insect (Fig. 5), which allows part of the H2 emitted from the anterior region (midgut, mixed segment, P1 and P3 segments) to diffuse across the epithelia into the adjacent posterior segments (P4a or P4b) containing efficient hydrogen sinks. Also formate, which was found in high concentration (>2 mM) in the hemolymph of Cubitermes orthognathus (Tholen and Brune, unpublished results) and which strongly stimulates the CH4 emission of isolated P3/4a and P4b sections (Table 2), has to be considered as a possible candidate for the transfer of reducing equivalents between the compartments. This is supported by results of Brauman et al. (Brauman et al., 1990; Rouland et al., 1993), who found hydrogen- and formate-utilizing methanogens to occur in similar numbers in gut homogenates of Cubitermes speciosus and other soil-feeding Termitinae.
The mixed segment and the third proctodeal segment (P3) are the major sources of H2 in the hindgut of the three soil-feeding Termitinae tested in this study. Luminal H2 partial pressures were in the same range as those in the hindgut paunch of Reticulitermes flavipes (Ebert and Brune, 1997). However, the anterior hindgut of soil- feeding Termitinae is characterized by an extremely high luminal pH, extending from the mixed segment to the P3, with an alkalinity maximum (pH 12) in the P1 compartment (Brune and Kühl, 1996), which also accumulates less H2 than the mixed segment or the P3. The P1 of Procubitermes aburiensis and Cubitermes umbratus contains a significantly lower density of microorganisms than all other hindgut compartments (Bignell et al., 1980; Tholen and Brune, unpublished results), and also O2 consumption in the alkaline gut regions has been attributed in part to the autoxidation of phenolic residues (Kappler and Brune, 1999). It is not yet clear which organisms or processes are responsible for the H2 production.
The large O2-consuming activities of all gut regions, the peripheral penetration of O2 into the hindgut lumen, and the anoxic status of the gut center are in agreement with previous results obtained for several other termites (Brune et al., 1995; Ebert and Brune, 1997; Kappler and Brune, 1999) and corroborate that also in soil feeders, a close juxtaposition of oxidative and fermentative processes within each hindgut dilation has to be expected. The increased H2 uptake rates of several hindgut compartments under oxic conditions may represent aerobic H2-oxidizing activities, but it is also possible that O2 affects the intestinal H2 production directly by its influence on the product pattern of the fermentative gut microbiota, as discussed by Ebert and Brune (Ebert and Brune, 1997). In that case, decreased H2 uptake rates under anoxic conditions would merely reflect a partial saturation of the H2-oxidizing processes by the increased internal H2 production.
It is likely that the methanogenic activities in the P3/4a compartment are restricted to the P4a segment, which contains microorganisms with green F420-like autofluorescence (Tholen and Brune, unpublished results). In contrast to the P4a, the P3 segment accumulated endogenous H2 to high concentrations (Fig. 3), but in view
of the high alkalinity (approx. pH 10) of the P3 segment in all soil-feeding Termitinae so far investigated (Brune and Kühl, 1996), it can be speculated that also H2- dependent acetogenesis will be restricted to the posterior, methanogenic gut region.
In those compartments, H2-dependent acetogens would have to compete with methanogens for H2. Even if methanogens prevent a luminal accumulation of H2 above the threshold values of homoacetogenic bacteria (Cord-Ruwisch et al., 1988) in the hindgut region posterior to the P3, cross-epithelial H2 transfer may still create potential microniches for homoacetogens situated within the hydrogen gradient, e.g., directly below the gut epithelium.
In that context, it seems important to recall that the hypothetical predominance of methanogenesis over reductive acetogenesis in soil-feeding termites is so far based only on the comparison of CH4 emission rates of living termites with the potential rates of H2-dependent acetogenesis in gut homogenates (Brauman et al., 1992). The in situ rates of reductive acetogenesis and the axial distribution of homoacetogenic bacteria remain to be determined. We have addressed these issues in a companion paper (Tholen and Brune, 1999).
Acknowledgments
This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) within the program 'Structural and Functional Analysis of Natural Microbial Communities'. We wish to thank Erika Banchio, Andrea Ebert, and Thorsten Lemke for their help with measurements, and Bernhard Schink for his continuing support.
We are indebted to several colleagues who helped collecting the termites species used in this study.
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