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Applied and Environmental Microbiology, October 2001, p. 4657-4661, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4657-4661.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cross-Epithelial Hydrogen Transfer from the Midgut
Compartment Drives Methanogenesis in the Hindgut of
Cockroaches
Thorsten
Lemke,1
Theo
van Alen,2
Johannes H. P.
Hackstein,2 and
Andreas
Brune1,*
Fachbereich Biologie, LS Mikrobielle
Ökologie, Universität Konstanz, 78457 Konstanz,
Germany,1 and Department of Evolutionary
Microbiology, Faculty of Science, Catholic University of Nijmegen,
NL 6525 ED Nijmegen, The Netherlands2
Received 10 May 2001/Accepted 1 August 2001
 |
ABSTRACT |
In the intestinal tracts of animals, methanogenesis from
CO2 and other C1 compounds strictly depends on
the supply of electron donors by fermenting bacteria, but sources and
sinks of reducing equivalents may be spatially separated. Microsensor
measurements in the intestinal tract of the omnivorous cockroach
Blaberus sp. showed that molecular hydrogen strongly
accumulated in the midgut (H2 partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior
hindgut. Moreover, living cockroaches emitted large quantities of
CH4 [105 ± 49 nmol (g of cockroach)
1
h1] but only traces of H2. In vitro
incubation of isolated gut compartments, however, revealed that the
midguts produced considerable amounts of H2, whereas
hindguts emitted only CH4 [106 ± 58 and 71 ± 50 nmol (g of cockroach)1 h1,
respectively]. When ligated midgut and hindgut segments were incubated
in the same vials, methane emission increased by 28% over that of
isolated hindguts, whereas only traces of H2 accumulated in
the headspace. Radial hydrogen profiles obtained under air enriched
with H2 (20 kPa) identified the hindgut as an efficient sink for externally supplied H2. A cross-epithelial
transfer of hydrogen from the midgut to the hindgut compartment was
clearly evidenced by the steep H2 concentration gradients
which developed when ligated fragments of midgut and hindgut were
placed on top of each other
a configuration that simulates the
situation in vivo. These findings emphasize that it is essential to
analyze the compartmentalization of the gut and the spatial
organization of its microbiota in order to understand the functional
interactions among different microbial populations during digestion.
| |
INTRODUCTION |
Methanogenesis is an
important electron sink in the intestinal tracts of terrestrial
arthropods such as Diplopoda (millipedes), Scarabaeidae (scarab beetles), Blattidae
(cockroaches), and Isoptera (termites) (12). It
has been postulated that termites contribute substantially to global
methane fluxes (9, 17, 26). According to recent estimates,
termites may account for about 4 to 10% of the global production of
this greenhouse gas (1, 19). The contribution of all
methane-producing arthropods together is likely to be much higher
(12, 13).
Methanogenic archaea in arthropods are generally restricted to the
hindgut, where they occur free-floating in the gut lumen, attached to
chitinous structures of the gut wall, or as intracellular symbionts of
gut-dwelling anaerobic protists (12). The most likely
electron donors for intestinal methanogens are
H2, formate, and methanol; there is no indication
that aceticlastic methanogenesis plays a major role. Hydrogen is the by
far most prominent among these potential substrates, particularly in
termites. All termites investigated to date are characterized by gut
segments with high H2 partial pressure (10,
20). In (phylogenetically) lower termites, which harbor large
numbers of protozoa in their hindguts (3), the
carbohydrate fermentation by anaerobic flagellates is likely to be the
major source of H2 (14, 16). In
(phylogenetically) higher termites, which possess a largely prokaryotic
gut microbiota and typically do not host any flagellates in their
hindgut, and also in other arthropods, the organisms and metabolic
pathways that are responsible for the formation of
H2 remain to be identified.
If methane-producing and hydrogen-consuming processes were
homogeneously distributed in the gut segments that emit methane, low
hydrogen partial pressures would be expected throughout the gut.
Notably, microsensor measurements in the methane-emitting hindgut of
the lower termite Reticulitermes flavipes have shown that
hydrogen accumulates to high partial pressures at the gut center,
whereas only small fluxes of hydrogen emanate from the hindgut
(10). This is due to the presence of significant
hydrogen-consuming activities at the periphery of the hindgut and
hydrogen-producing activities apparently prevailing in the central
region. In other words, the spatial organization of the
hydrogen-producing and hydrogen-consuming microbiota controls hydrogen
metabolism and methanogenesis in the hindgut of lower termites
(5, 10, 24).
In the highly differentiated intestinal tract of higher termites
(Cubitermes spp.), hydrogen-emitting and methane-producing gut compartments can be discriminated. The latter exhibit a significant hydrogen uptake activity when provided with external hydrogen (20). Based on the proximity of the hydrogen-producing and
the hydrogen-consuming, methane-producing gut segments in vivo, a cross-epithelial hydrogen transfer has been postulated (6, 20). Since a striking compartmentalization of the intestinal tract is also present in other methane-producing arthropods
(8), it can be assumed that this phenomenon is not
restricted to higher termites. Here we describe the cross-epithelial
transfer of hydrogen in Blaberus sp., a large, omnivorous
cockroach with a highly differentiated intestinal tract.
| |
MATERIALS AND METHODS |
Organisms.
Cockroaches (Blaberus sp.) were bred
at the University of Nijmegen. They were fed a commercial pelleted
rabbit diet, supplemented by apples, raw potatoes, and water ad
libitum. Only adult cockroaches were used for the experiments.
For measurements with isolated guts or ligated gut segments,
cockroaches were anesthetized with
N2-CO2 (80:20, vol/vol),
decapitated, and dissected in Ringer's solution (7) to
prevent dehydration of the gut. When midgut and hindgut were incubated
separately, each gut segment was ligated at both ends with thin cotton
or nylon thread using a pair of watchmaker's forceps and a stereomicroscope.
CH4 and H2 production rates.
Living
cockroaches were placed in 250-ml glass bottles, which were sealed with
butyl-rubber stoppers, and were incubated for several hours under air
at room temperature. Ethane (1 ml) was added as an internal standard
(11). At regular intervals, gas samples (200 µl) were
taken using hypodermic needles and 1-ml syringes and analyzed for
methane and hydrogen by gas chromatography (21, 22). As a
rule, gases were measured at 3 h and 17 to 22 h after the
start of the incubation.
Gut segments were incubated in 2 ml of HEPES buffer (50 mM HEPES, 50 mM
NaCl, 5 mM Na
2HPO
4, 1.5 mM
KH
2PO
4 [pH 7.0]) in 10-ml
vials (
21). Since pilot experiments had shown that under
these
conditions hydrogen production was linear for up to 29 h and
the
production of methane from endogenous substrates was linear for
up
to 48 h, gas production rates were analyzed using the same
protocol as for living
cockroaches.
Completely separated midgut and hindgut segments incubated in the same
vial did not differ significantly with respect to gas
production from
intact guts merely ligated between midgut and
hindgut; therefore, the
data for all coincubations were pooled.
Since moderate shaking of the
vials did not stimulate the production
of methane and hydrogen by
isolated guts or gut segments, we concluded
that gas diffusion from the
buffer into the headspace was not
limiting under the experimental
conditions.
Hydrogen microsensor measurements.
Hydrogen concentration
profiles were measured with polarographic H2
microsensors, which had basically the same design as Clark-type O2 microsensors (18) and were
modified according to Witty (25). The microsensors were
constructed in our laboratory in Konstanz and were tested and
calibrated as described earlier (10). The detection limit
for H2 was about 100 Pa; the stirring sensitivity was always <1% of the signal obtained at a H2
partial pressure of 20 kPa (calibration gas,
N2-H2, 80:20 [vol/vol]).
For profile measurements, the microsensors were mounted on a manual
micromanipulator (10). Each set of experiments was
reproduced with at least four different animals; the profiles shown in
the figures represent typical examples.
For axial hydrogen profiles, cockroaches were dissected and the intact,
fully extended gut was placed in an incubation chamber
(16 by 16 mm,
100 mm long) and fixed with minutia pins on a thin
silicone layer at
the bottom of the chamber. The chamber was filled
with air-saturated
Ringer's solution and irrigated at a continuous
flow rate (5 ml
min
1). Under these experimental conditions, the
dissected guts exhibited
a moderate peristalsis that persisted for
several hours, indicating
that they were still physiologically
active.
For radial hydrogen profiles, shorter, ligated sections of midgut or
hindgut were embedded in a smaller chamber in Ringer's
solution
solidified with 0.5% agarose and incubated under a controlled
gas
headspace. The agarose layer above the gut did not exceed
2 mm to avoid
the formation of a diffusion barrier. The chamber
was continuously
flushed with the desired gas mixture. To demonstrate
cross-epithelial
transfer of hydrogen, ligated sections of midgut
and hindgut that were
lying in proximity in vivo (Fig.
1) were
positioned in direct contact with each other and embedded in Ringer's
solution solidified with 0.5% agarose. The experimental setup
was the
same as for the other radial profiles; the gut sections
were incubated
under air. The details of the experimental setup
have been described
earlier (
10).
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FIG. 1.
Semischematic presentation of the intestinal tract of an
adult Blaberus sp. cockroach, illustrating the intimate
intertwining of midgut (M) and hindgut (H) in the abdomen. Open (M) and
closed (H) arrows indicate the direction of flow from crop (C) to
rectum (R). The coiling of the individual loops in vivo is even
tighter, especially in the larvae.
|
|
All hydrogen concentrations are reported as partial pressures. At
atmospheric pressure, a partial pressure of 1 kPa equals
a mixing ratio
of 1% H
2. At this partial pressure, pure water
dissolves about 8.0 µmol of H
2 per liter
(20°C). However, we did
not convert H
2 partial
pressures to molarities, since this would
require knowledge of the
exact solubility coefficient(s) for H
2 in
different gut
contents.
| |
RESULTS |
Axial hydrogen profiles and emission rates of H2 and
CH4.
Axial hydrogen profiles of the intact guts of
Blaberus sp. revealed large differences among the various
gut segments (Fig. 2). While hydrogen
partial pressures were always below the detection limit of the
microsensor (0.1 kPa) in the posterior hindgut of all animals
investigated, the midgut segments accumulated substantial amounts of
H2. Hydrogen profiles of the midgut varied
considerably among individual animals (data not shown), ranging from 3 to 26 kPa H2. The average partial pressures of
hydrogen in the anterior, median, and posterior regions of the midgut
were 17, 6, and 11 kPa (n = 7), respectively. In most
cases, the accumulation of hydrogen extended into the anterior hindgut.
However, hydrogen accumulation was never observed in the posterior part
of the hindgut or the crop.
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FIG. 2.
Typical axial hydrogen profile ( ) of an intact, fully
extended gut of Blaberus sp. and average H2
partial pressures (×) in the anterior, median, and posterior midguts
of different animals (means ± standard deviations;
n = 7). Hydrogen was measured with a microsensor;
all readings were taken at the gut center.
|
|
Measurement of hydrogen and methane emissions by intact, living
cockroaches (Fig.
3) showed that the
animals emitted only
traces of H
2 but large
quantities of CH
4 [105 ± 49 nmol (g of
cockroach)
1 h
1
(
n = 12)]. Notably, isolated midguts incubated in
vitro emitted
significant amounts of H
2 at
constant rates [106 ± 58 nmol (g
of
cockroach)
1 h
1],
whereas isolated hindguts emitted only CH
4
[71 ± 50 nmol (g
of cockroach)
1
h
1]. When intact intestinal tracts with
ligated midgut and hindgut
segments were incubated in the same vial,
methane emission increased
over that of isolated hindguts [91 ± 26 nmol (g of cockroach)
1
h
1], whereas the hydrogen concentrations in
the headspace remained
low (Fig.
3).
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FIG. 3.
Hydrogen and methane emission rates of whole insects,
intact guts, and ligated midgut and hindgut segments of
Blaberus sp. Values are means (plus standard deviations)
of 12 different assays each. The large variance is due to the
individual differences among the animals.
|
|
Radial hydrogen profiles.
Isolated, agarose-embedded midguts
showed the highest hydrogen accumulation at the center of the gut (Fig.
4A). In the gut periphery,
H2 diffused across the gut epithelium into the
surrounding agarose. When midgut sections were incubated under a
headspace of air containing 20 kPa of H2, the
hydrogen concentration in the midgut lumen increased, and the shape of
hydrogen profiles did not show any evidence of hydrogen uptake
activities in the midgut periphery. Isolated hindgut sections, on the
other hand, showed no detectable hydrogen accumulation, confirming the
results of the axial profiles (Fig. 4B). Notably, all hindgut sections tested (n = 12) were efficient hydrogen sinks when
incubated under air containing 20 kPa of H2.
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FIG. 4.
Typical radial hydrogen profiles of agarose-embedded
midgut (A) and hindgut (B) sections of Blaberus sp.
incubated under a gas headspace of air () or air enriched with 20 kPa of H2 ( ). Hydrogen was measured with a
microsensor.
|
|
To test whether hydrogen is transferred across the gut epithelium from
midgut to hindgut, short, ligated sections of midgut
and hindgut that
were also in direct contact in vivo (Fig.
1)
were placed on top of each
other and embedded in agarose. Radial
hydrogen profiles revealed an
accumulation of hydrogen at the
center of the midgut sections and a
diffusion of hydrogen into
the surrounding agarose. Towards the hindgut
sections, however,
steep diffusive gradients of hydrogen developed
(Fig.
5). Despite
its high accumulation
in the midgut, hydrogen was completely consumed
within the first 100 µm beyond the hindgut epithelium. In the
central portion of the
hindgut lumen, there was no evidence of
any accumulation of hydrogen.
Comparable profiles were obtained
irrespective of the positioning of
the gut sections, i.e., whether
the midgut was placed on top of hindgut
or vice versa (data not
shown). Steep hydrogen gradients developed
across the midgut-hindgut
interface of all gut sections which were
tested in this experimental
setup (
n = 6).
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FIG. 5.
Typical radial hydrogen profile through ligated midgut
and hindgut sections placed in direct contact with each other and
embedded in agarose, mimicking the in vivo arrangement of the sections
(Fig. 1). Guts were incubated under air, and hydrogen was measured with
a microsensor.
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|
| |
DISCUSSION |
The strong accumulation of hydrogen in the midgut of
Blaberus sp. and its emission from isolated midgut segments
contrasts strongly with the nearly complete absence of
H2 in the breath of cockroaches. This implies
that almost all H2 produced in the midgut is
consumed inside the animal. Considering the anatomy of the intestinal
tract and the juxtaposition of hydrogen-producing and
hydrogen-consuming gut segments in vivo (Fig. 1), it is tempting to
assume that the hydrogen formed in the midgut diffuses across the gut
epithelia into the hindgut, where it is removed by a hydrogenotrophic microbiota. Such a model has already been proposed to explain a similar
phenomenon in soil-feeding termites (20) and is supported by the stimulation of methane emission by externally applied hydrogen observed in living termites (10, 20) and cockroaches
(J. H. P. Hackstein and T. van Alen, unpublished results).
This is the first time, however, that the cross-epithelial transfer of
endogenously produced hydrogen was measured directly. Coincubation
experiments and radial hydrogen concentration profiles of isolated
midgut and hindgut segments of Blaberus sp. clearly identify
the presence of a strong hydrogen source and the absence or
insignificance of hydrogen-consuming processes in the midgut and the
presence of a highly efficient hydrogen sink in the hindgut. The
cross-epithelial transfer of hydrogen is clearly evidenced by the
radial hydrogen profiles obtained when ligated fragments of midgut and
hindgut were placed on top of each other, a configuration that
simulates the in vivo situation (Fig. 1).
Notably, H2 did not accumulate in the headspace
when midgut and hindgut were coincubated in vitro, whereas the
methane production increased considerably (Fig. 3). This
observation strongly argues for a preferential stimulation of
methanogenesis by cross-epithelial hydrogen transfer. The hydrogen
emission rates of isolated midguts are about 5.5-fold higher than the
corresponding stimulation of methanogenesis during coincubation.
Assuming that H2 consumed by the hindgut is used
exclusively for methanogenesis from CO2, a 4:1
stoichiometry would be expected. Therefore, it is possible that other
hydrogen-consuming processes, e.g., reductive acetogenesis, are
stimulated as well. The large variation in the emission rates between
individual cockroaches, which also depends on developmental stage, sex,
and diet (15) (unpublished observations), does not allow a
definite conclusion based on this data set. Nevertheless, the
stimulation of reductive acetogenesis by exogenous
H2 has been clearly demonstrated in the hindgut
of soil-feeding termites with radiotracer techniques (23).
It should be pointed out that there is so far no satisfactory
explanation for the fate of the hydrogen which apparently emanates from
the side of the midgut which is not in contact with the hindgut in situ
(Fig. 5). Most likely, oxygen supply to the gut, which is effected by
the tracheal system in the living cockroach (a combination of advective
and diffusive transport in the gas phase and only a short-range
diffusive transport in the aqueous phase), is restricted by embedding
the gut in agarose. The resulting diffusion limitation would create a
significant oxygen deficit, which would stimulate the formation of
H2 in the midgut (10). Therefore, it
is possible that in vivo fluxes of H2 from the
midgut are lower than those determined under experimental
conditionsor that intercompartmental transfer of
H2 is not the only reason for the absence of
hydrogen emission in living cockroaches.
The presence of highly differentiated gut segments in a variety of
methane-producing arthropods (12) suggests that a
cross-epithelial transfer of reducing equivalents between different gut
compartments is likely to occur in other animals as well. The diffusion
of hydrogen is facilitated by the inherent permeability of the
intestinal epithelia to gases; transepithelial gas exchange between gut
and bloodstream (or between gut and tracheal system) is well
established in humans (4) and in a number of arthropods
(2). Nevertheless, it should be considered that formate
and methanol are also potential substrates for methanogenesis in the
hindgut. The hemolymph of soil-feeding termites, for example, contains
appreciable concentrations of formate (23). Formate seems
to be produced by microbial fermentations in the midgut and
stimulates methanogenesis in the hindgut (20). A similar
situation is present in the larvae of scarab beetles (Pachnoda sp.) (T. Lemke and A. Brune, unpublished results).
Moreover, methanol is formed as the demethylation product of pectins in the midgut of several species of cockroaches and the larvae of Pachnoda (21; J. H. P. Hackstein,
J. A. de Gouw, and C. Warneke, unpublished results). It remains to
be shown that methanol is transported between the gut compartments, but
there is evidence that Methanomicrococcus blatticola, an
obligately methanol-reducing, methanogenic bacterium, is a major
methanogenic organism in the hindgut of the cockroach Periplaneta
americana (21). While methanol should be able to pass
through the epithelial barrier easily, formate and other charged
metabolites would require specific transport systems.
The results of the present study emphasize that we are just beginning
to understand the interdependence of microbial processes in arthropod
guts. Any such gut, no matter how small it is or how simple it seems to
be at first glance, is a complex and highly structured environment
(6). In order to understand the physiology of the
digestive tract and to resolve the functional interactions among the
different microbial populations, it is essential to analyze the
compartmentalization of the gut and the spatial organization of its
microbiota in more detail.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fachbereich
Biologie, LS Mikrobielle Ökologie, Universität Konstanz,
Fach M 654, 78457 Konstanz, Germany. Phone: 49-7531-883282. Fax:
49-7531-882966. E-mail: Andreas.Brune{at}uni-konstanz.de.
| |
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Applied and Environmental Microbiology, October 2001, p. 4657-4661, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4657-4661.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
