Hydrogen profiles and localization of methanogenic activities in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.)

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 H(2) partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H(2). Posterior to the P3 segment, however, H(2) concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H(2) was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H(2) or formate was added. Addition of H(2) to the gas headspace stimulated CH(4) emission of living termites, indicating that endogenous H(2) production limits methanogenesis also in vivo. At the low H(2) 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 H(2) 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 H(2)-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497-4505, 1999).

FIG. 1

Gut morphology of a Cubitermes sp. worker termite, also representative of other soil-feeding species of the subfamily Termitinae. The gut was drawn in its unraveled state to illustrate the various segments: C, crop; M, midgut; ms, mixed segment; P1 to -5, proctodeal segments 1 to 5, respectively. The average luminal pH was determined for the indicated gut regions in Cubitermes speciosus by using intact guts and glass pH microelectrodes (12). When gut sections were used, the guts were separated at the indicated positions.

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 (●) 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 definitions of abbreviations.

FIG. 3

Radial profiles of the oxygen (A) and hydrogen partial pressure (B and 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 mark represents a distance of 250 μm. Oxygen profiles (A) were measured under an air headspace, and hydrogen profiles were measured under air (●) or under N₂ (○), without (B) or with (C) the addition of H₂ (20 kPa) to the headspace gas. In the case of the mixed segment (ms), an N₂ headspace with 5 kPa of H₂ was also used (□). The graphs represent typical sets of similar profiles obtained in numerous experiments with different guts.

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 (± standard deviations) of three to five different profiles.

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 the legend to Fig. 1.