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Atmospheric carbon dioxide and acetogenesis in the termite Nasutitermes walkeri (Hill)
Cony. Biochem. Physiol. Vol. 107A, No. I, pp. 113-I 18, 1994 Printed in Great Britain
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0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
Atmospheric carbon dioxide and acetogenesis in the termite Nasutitermes walkeri (Hill) C. M. Williams, P. C. Veivers, M. Slaytor and S. V. Cleland Department
of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia
CO, concentration inside Nasutiiermes walkeri nests is higher than in the external atmosphere. At concentrations up to 2%, CO* absorption by the termite is dependent on atmospheric CO1 concentration. Atmospheric CO, is reduced in uiuo to acetate in the termite but the amount of acetate in the termite is constant and is not related to the atmospheric CO2 concentration. Key words: Nasutitermes walkeri; Termites; Acetogenesis; Carbon dioxide
Comp. Biochem. Physiol. 107A, 113-118, 1994.
Introduction A CO* concentration higher than normal atmospheric is characteristic of nests of social insects (Nicolas and Sillans, 1989). Within the Isoptera the concentration is usually quite high and subject to both diurnal and seasonal fluctuations. For example, diurnal fluctuations in atmospheric CO2 concentration between 1.Oand 1.89% have been measured in Nasutitermes exitiosus (Day, 1938). Similarly, diurnal variations of O&2.9% (Liischer, 1961) and seasonal variations of 1.2-5.2% (Matsumoto, 1977) have been recorded within nests of the termite subfamily Macrotermitinae. Within this subfamily, Macrotermes natalensis passively controls CO2 concentration through a nest design that facilitates air circulation, but will rebuild porous parts of the nest if the internal COZ concentration increases from 1 to 2% (Ruelle, 1964). Many effects of COZ on insects have been studied including some on biochemical processes (Nicolas and Sillans, 1989). Carbon dioxide has one important role in the anaerobic metabolism of gut microbes in the paunch of termites: it acts as an electron sink for reducing power generated in the paunch (Breznak and Switzer, 1986). Reoxidation of reducing power appears to be directed towards acetogenesis rather than methanogenesis or hydrogen production as in the rumen. Thus one possible fo: M. Slaytor, Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia. Fax: 02-692-4726. Received 2 March 1993; accepted 31 March 1993. Correspondence
role for elevated atmospheric CO, in termite nests could be to ensure that acetogenesis proceeds at an optimal rate. In this paper we present data from in uivo experiments which show that atmospheric CO, can be reduced to acetate in the termite and that the amount of CO2 reduced is proportional to the atmospheric concentration but that the overall rate of acetogenesis in the termite is independent of the CO* concentration in the atmosphere.
Materials and Methods Termites Worker caste termites of Nasutitermes walkeri (Hill) collected from galleries leading to arboreal nests in suburban Sydney were used within 4 hr of collection except where otherwise indicated. Based on the weight of 100 worker caste termites, the average weight of freshly collected termites used was 8.5 mg. The paunch volume was measured by noting the increase in volume when 40 paunches were added to 0.5 ml of buffer and by measuring the decrease in weight of 10 paunches when freeze-dried, using 10 replicates. The haemolymph volume was estimated by injecting 120 nl of [‘4C]polyethylene glycol into 10 termites using the method of Wharton et al. (1965). In situ CO;! concentration in termitaria and atmospheric CO2 Air samples from within the termitaria were taken with a gas probe or, from galleries, with syringes. The arboreal nests protruded from the
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C. M. Williams
114 tree trunk extension
between 30 and 40 cm with a vertical of 70-80 cm and were situated at
heights between 15 and 20 m. The probe was constructed from two pieces of stainless steel tubing, an outer casing (diameter 2.5 mm) enclosed a sliding insert (diameter 2.3 mm) which was secured to a needle the tip of which was covered with Parafilm’@ to prevent blockage during insertion into the nest. After the probe had been inserted to a depth indicated by markers on the outer tube, it was withdrawn slightly and the insert extended to displace the Parafilm@. Air samples were collected from the nest in 10 ml plastic syringes or from galleries in 5 ml plastic syringes. Syringes were then sealed with the needle embedded in a rubber stopper. The concentration of CO2 in 5 ml aliquots was measured by gas chromatography within 2 hr of collection using a Porapak QS column (Alltech, Deerfield, IL, U.S.A.) in a Gow Mac gas chromatograph (model 69-150). The flow rate of the helium carrier was 60 ml/min-‘, the column inlet pressure 38 kPa and the bridge current 200 mA. An Autolab Integrator (Model 23000010, Spectra Physics, CA, U.S.A.) was attached for the calculation of peak areas. Industrial grade CO2 was used as a standard. The concentration of atmospheric CO* was similarly measured.
RQ, H, and CH, estimations
Termites (3 x 100) were placed in Quickfit B14 Erlenmeyer flasks (25 ml) sealed with Subaseals and 200 ~1 or 5 ml aliquots of the atmospheric gas were removed as required for CO, analysis or O,, H, and CH, analysis, respectively. The procedure was repeated 15 times. Aliquots (5 ml) removed for 02, Hz and CH4 analysis were replaced with 5 ml of air. The aliquots were analysed at room temperature on a copper column 3 m x 6 mm) packed with a molecular sieve 5 6 60/80 (Alltech, Deerfleld, IL, U.S.A.) in a Hewlett Packard gas chromatograph (model 5710) with a thermal conductivity detector. The following conditions were used: carrier gas, argon; flow rate, 20 ml/min-‘; column temperature, 150°C; detector temperature, 100°C; sample loop, 5 ml. The chromatograph was linked to a Hewlett Packard integrator and the column was calibrated with appropriate standards. 200 ~1 aliquots removed for COZ measurement for RQ estimations were analysed at room temperature on a Porapak QS (Alltech) column in a Gow Mac (model 69-150) gas chromatograph fitted with a thermal conductivity device. Bridge current, 200 mA; carrier gas, helium, flow rate, 60 ml/min-‘. The
et al.
concentration of CO* was calculated from peak heights and was linear over the range used (up to 10%). O2 utilization under non -atmospheric concen trations of CO2 One hundred worker caste termites were placed in Quickfit B14 Erlenmeyer flasks (25 ml) which were then fitted with Subaseals. COZ atmospheres were established with industrial grade CO*. Gas samples (200 ~1) were taken, over a 5 hr incubation at 25°C and analysed for O2 by injecting into a copper column (3 m x 6 mm) packed with a molecular sieve 5 8, 60/80 (Alltech) in a Gow Mac gas chromatograph (model 69-150). The flow rate of the helium carrier was 60ml/min-‘, the column inlet pressure 38 kPa and the bridge current 200 mA. An Autolab Integrator (Model 23000010, Spectra Physics, CA, U.S.A.) was attached for the calculation of peak areas. Ambient air (20.946% 0,) was used as a standard. 14C02 absorption from atmospheric
and [14C]acetate 14C02
production
was prepared from NaH14C03 ‘TO2 (0.1 mCi/mmol; Amersham International, U.K.) or Ba14C03 (51 mCi/mmol; Amersham). Different partial pressures of CO* were established in stoppered 25 ml Erylenmyer flasks containing 50 termites by injection of 14C02. Termites were either incubated for 1 hr at 25°C under varying 14C02 atmospheres or were incubated at 25°C for varying periods of time at constant CO* (2%). After incubation, termites were probe-sonicated (4°C for 30 set at 40 W; Branson model B 12 sonicator) in 2 M NaOH (600 ,ul) and then centrifuged (15 OOOg for 10 min at 4°C); aliquots were used to determine the total amount of 14C-labelled material in the termite. Aliquots (200 ~1) were placed in the outer well of a Warburg flask (30 ml) and the 14C02 liberated with 500~1 2 M HCI was trapped over 4 hr with 300 ~1 of freshly prepared 2 M NaOH in the centre well. Aliquots (100 or 200 ~1) from the outer well were used for HPLC analysis, enzymatic determination of CH,COOH (Boehringer Mannheim kit no. 148 261) and scintillation counting to determine incorporated 14C02. Aliquots from the inner well were used to estimate “C02. Aliquots (100 ,ul) for scintillation counting were applied to glass microfibre filters (2.1 cm; Whatman International Ltd, U.K.) impregnated with 100 ~1 of freshly prepared 10% Ba(CH,COO), . Dried filters were placed in 20 ml glass scintillation vials containing 5 ml of scintillation fluid (0.6% PPO in toluene).
CO2 and acetogenesis in termite
HPLC The acidified homogenate from the outer well was filtered through a 0.45 pm Millipore filter and injected through a Rheodyne 7125 syringe loading sample injector using 25-200 ,ul syringes (SGE, Melbourne) into an HPLC apparatus which consisted of an LKB Liquid Chromatograph containing the following modules: a Model 2152 HPLC controller, a 2150 pump and a 2040-203 gradient mixing valve. Absorbance was monitored at 200 nm with a 2151 variable wavelength monitor and data were recorded and integrated by the Maxima 820 Chromatography work station (Dynamic Solutions). A Bio-Rad Aminex HPX-87H Organic Acid Analysis (cation-exchange) column (300 x 7.8 mm), fitted with a 5 cm H+ guard column (Bio-Rad 1250129; 40 x 4.6 mm) containing the same resin was used. Samples were eluted isocratically with 5 mM HzS04 (flow rate 0.5 ml/mine’) at 45°C. Fractions eluting from the column were collected, on the basis of retention time, dropwise into scintillation vials, using a Gilson Fraction Collector (Model 202). The metabolites were identified by comparison with retention times of standards. The distribution of 14C0, in metabolites was determined by the level of radioactivity in collected fractions. The pH was adjusted to 8-9 by addition of 2.5 M KOH and 0.63 M KHC03 (10-100 ,ul depending on fraction volume). All fractions were dried overnight at 7o”C, redissolved in 100 ,~l of milli-Q water and 5 ml of scintillation fluid (Optiphase “HiSafe” 3; LKB Scintillation Products) added. Scintillation counting Radioactivity was measured either in a Beckman (model LS 3800) or in a Wallac (model 1410; Pharmacia) liquid scintillation counter. Samples processed by the Beckman counter were corrected for quenching using NaH14C0, as a standard; the Pharmacia counter was programmed to correct for quenching and chemoluminesence automatically. Standards for 14C0, were prepared by absorbing aliquots of the experimental gas mixture in Soluene@.
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at depths of 10 and 15 cm analysed for mean values of 0.48 f 0.08% (n = 2) and 0.68 + 0.03% CO, (n = 2), respectively. It was not possible to probe beyond 20cm without bending the probe. The fragility of the galleries made gas sampling difficult but mean values of 0.14 + 0.03% (n = 4) were recorded from intact galleries from two different nests on three separate days. Same day samples (n = 2) from one nest indicated mean concentrations of 0.14 * 0.01% co,. When freshly collected worker caste termites of N. walkeri were incubated in air at 25°C they consumed O2 and produced COZ at 82 + 2 and 87 It 2 nmol termite-’ hr-’ respectively. Hydrogen was produced at 0.74 + 0.05 nmol termite-’ hr-‘. The amount of CH4 produced (N 1 nmol termite-’ hr-’ ) was on the lower limit of detection. The RQ value for all determinations was in the range 1.1 + 0.14. O2 utilization slowly decreased at higher CO, concentrations from a rate of 83 + 5 nmol O2 termite-’ hr-’ (n = 6) at normal atmospheric CO2 concentration though, at atmospheres of 2% COZ the utilization was not significantly altered (80 k 13 nmol O2 termite-’ hr-’ (n = 2) (Fig. 1). At CO2 concentrations higher than shown in Fig. 1, the rate of 0, utilization decreased dramatically. It declined to 41 k 7 (n = 3) and 12 & 6 (n = 2) nmol termite-’ hr-’ at CO, concentrations of 75 and 90%, respectively. Termites appeared to be normally active during incubation at concentrations of CO2 less than or equal to 5% but ceased moving within 1 min of being exposed to CO* concentrations of 75% and greater. Incubating termites in 14C02 atmospheres containing increasing amounts of total CO2 showed that the amount of “C-labelled material found in the termite was linear between 0.5 and 2% atmospheric CO2 and that the rate of uptake was 26 nmol termite-’ hr-’ %-’ CO*. At higher concentrations (up to 5%) the rate decreased (Fig. 2). Thus, 2% CO2 appeared to be the CO2
Statistical analysis All errors are represented by the standard error of the mean. Correlation coefficients for calculated rates are expressed as r.
Rl?SUltS
501
’ 0
Nasutitermes walkeri is common in the Sydney region and the galleries on the bark connecting the nest with the ground allow for multiple and rapid collection of workers. Gas samples for CO, estimation, collected from two nests,
1
2
3
4
5
Atmospheric CO2 (%) Fig. 1. Oxygen utilization as a function of atmospheric CO2 concentration in N. walkeri. The 0 sample is at normal atmospheric CO, concentration (0.03%).
C. M. Williams et al.
0
0
1
2
3
4
5
Atmospheric Ct& (%)
20
60
40 Time (min)
Fig. 4. Uptake of CO, by N. w&eri as from an atmosphere containing 2% CO,.
Fig. 2. Uptake of CO, by N. wnlkeri as a function of atmospheric CO, concentration.
over 60min. In two experiments, the rate of [14C]acetate production over 1 hr from absorbed concentration at which absorption of 14C02 is 14C02 at 2% atmospheric CO2 was 1.77 optimal and was used for most incubations. (r = 0.99) and 2.80 (r = 0.98) nmol terWhen termites were incubated at 0.7% CO*, mite-’ hr-‘; this corresponds to rates of CO* 60% of the absorbed radioactivity was in the incorporation of 3.5 and 5.6 nmol CO2 tertermite body, the remainder being in the gut mite-’ hr-‘. The incorporation of 14C02 into (Fig. 3). At least 91% of the “C-labelled ma- [14C]acetate over the first 10min in the first of terial was 14C02. At 2% the absorption of 14C02 these experiments, when the rate of incorporwas linear over the first 30 min (rate 0.64 nmol ation is rapidly changing, is shown in Fig. 5. 14C02 termite-’ min-’ ) but appeared to de- Acetate was the main metabolite formed from crease over the next 30 min (0.23 nmol 14C0, 14C0, on the basis of HPLC data. Two minor termite-‘min-‘) (Fig. 4). The experiment was metabolites were not identified: formate, propirepeated: the trends were similar, although the onate, butyrate, lactate and pyruvate were elimrate of 14C0, absorption in the second experinated as possibilities. The total amount of iment was approximately twice that of the first. acetate in whole termites in these experiments At least 9% of the absorbed 14C02 was incorwas 13.9 kO.4 (n = 8) and 19.3 kO.2 (n = 6) porated into metabolites, corresponding to rates nmol acetate/termite. The rate of acetogenesis of COz incorporation of 3.2 and 6.4 nmol ter- as a function of atmospheric CO, did not appear mite-’ hr-‘, respectively, for the two experto increase beyond atmospheres of greater than iments. The rate of 14C02 incorporation into 3% (Fig. 6). metabolites at 2% atmospheric CO* was linear The paunch of N. walkeri has a volume of 1.5 + 0.2 ~1 (n = 4) using the volumetric method and 1.49 f 0.08 ~1 (n = 10) using the freeze drying method, and contains 12 + 1 nmol of acetate (n = 15); the concentration is thus approximately 8 mM. The haemolymph volume was g f 1.5 + 0.1 ,ul (n = 10) and the acetate concentration was 5 mM. 2 sg g 20-
3o
-
lo0
0
1
2
3
Time (h)
Fig. 3. Uptake of CO, into the gut and body of N. walked. l gut; 0 body. Termites were incubated for 3 hr in the presence of 0.7% CO, prepared as described in the text. Samples were taken at the times indicated. Ten termites were degutted and the guts and bodies mashed and placed overnight in scintillation vials at 50°C containing Soluene& (1 ml). After cooling, scintillation fluid was added and radioactivity estimated in a Beckman (mode1 Is 3800) scintillation counter. Samples were corrected for quenching using [U-‘4C]glucose as standard.
0
2
4
6
8
10
Time (min) Fig. 5. Acetopnesis in N. walkeri from atmospheric CO,; concentration of COr in atmosphere, 2%.
CO, and acetogenesis in termite
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Cubitermes speciosus acetogenesis is a minor process compared to methanogenesis (Brauman et al., 1990). Earlier gloomy predictions of global catastrophe due to overproduction of CH, by termite activity have fortunately not occurred (Rasmussen and Khalil, 1983) but methanogenesis is clearly an important process in many termite genera (Seiler et al., 1984). Our in vivo results clearly show that atmospheric CO2 can be reduced to acetate. As the overall 0 1 2 3 4 5 rate of acetogenesis does not increase with increasing CO2 concentrations, the atmospheric Atmospheric CO,(%) CO, presumably exchanges with that in the Fig. 6. Rate of acetate formation in N. walkeri from body and the paunch. This exchange is quite atmospheric CO, as a function of atmospheric CO,. rapid as acetate derived from atmospheric CO* can be detected within 2 min and that within 10 min the rate of production is 50% that of the maximum rate. Discussion On the calculation that glucose is the only The concentration of CO2 in both the nest carbohydrate being assimilated, a reasonable and the galleries of Nasutitermes walkeri is assumption as 85% of carbohydrate metabsignificantly higher than that of air though the olised in R. flavipes is glucose (Odelson and Breznak, 1983), then the rate of 0, utilization concentration in the nest is lower than that reported for N. exitiosus (Day, 1938) and other indicates that glucose is being metabolized in N. mound builders. Nest COr measurements on walkeri at about 14 nmol termite-’ hr-‘. In R. N. walkeri were made on early morning j7avipes, one-third of acetate production is as a samples, known to be the lowest diurnal level result of CO2 reduction (Breznak and Switzer, 1986). Hence it is reasonable to suppose that in in N. exitiosus (Day, 1938). Nasutitermes N. walkeri, acetogenesis from CO, reduction is walkeri is the first arboreal nest building termite on which CO* measurements have been proceeding at 14 nmol termite-’ hr-‘. Our results show that about 10% of this can come recorded. The increasing CO, gradient towards the centre of the nest in N. walkeri was also in vivo from atmospheric CO, when the atmosfound in N. exitiosus (Day, 1938) and may be pheric CO* concentration is 2%. On the basis of O2 utilization, the overall rate related to the distribution of termites within the of metabolism is hardly affected by increasing nest. atmospheric CO, concentration which also does The RQ values for freshly collected termites, though variable over a comparatively wide not affect the total amount of acetate in the range, appear centred on 1.0 indicating that termite. A maximum rate of acetogenesis of only carbohydrate, presumably cellulose, and 0.6 nmol termite-’ hr-’ from atmospheric COz not lignin is being digested (Odelson and Brez- occurs in atmospheres of about 3% CO*, As nak, 1983). The moderate decline in 0, utilis- this rate is maintained, but not exceeded, at ation over the physiological CO, atmospheric higher CO, atmospheres it presumably repconcentrations tested is consistent with the well- resents a steady state. In vitro experiments with documented anaesthetic effects of CO2 in insects gut homogenates of R. flavipes indicate that (Nicolas and Sillans, 1989). The small amounts Hz can be used for acetogenesis and that acetoof H, and CH, evolved suggest that most of the genesis is dependent on Hz concentration reducing power produced in the anaerobic (Breznak and Switzer, 1986). A H&O,-utilizpaunch is used for acetogenesis from CO* as it ing acetogen has been isolated from the gut of is in other nasutes such as N. costalis and N. N. nigriceps (Breznak et al., 1988). These expernigriceps (Breznak and Switzer, 1986). On a iments, however, do not show that H, is the molar basis, the evolution of H, or CH, was in vivo reducing agent. The absence of for-mate about 1% that of O2 consumption or CO2 or propionate as in vivo reduction products of production as has also been reported in the atmospheric CO2 in N. walkeri is in contrast to lower termite Reticulitermes flavipes (Odelson in vitro results obtained from gut homogenates and Breznak, 1983). Not all termites are as of N. costalis and N. nigriceps (Breznak and economical with the available reducing power Switzer, 1986). from fermentation and squander it in CHI proOur major conclusion is that although duction. For example, Zootermoposis angustiatmospheric CO2 can be reduced to acetate collis produces 10.3 nmol termite-’ hr-’ of CH, in vivo in N. walkeri, this process is not (Messer and Lee, 1989) and in the soil-feeder needed to achieve a maximum rate of
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acetogenesis and thus a biochemical explanation for the high CO, concentration in the nest remains unanswered. Acknowledgemenrs-This work was supported in part by a grant from the Australian Research Council.
References Brauman A., Labat M. and Garcia J. L. (1990) Preliminary studies on the gut microbiota of the soil feeding termite: Cubitermes speciosus. In Microbiology in Poecilotherms (Edited by L&e-l R.), pp. 73-77. Elsevier, Amsterdam. Breznak J. A. and Swit& J. M. (1986) Acetate synthesis from H, plus CO, by termite gut microbes. Appl. environ. Microbial. 52, 623630.
Breznak J. A., Switzer J. M. and Seitz H.-J. (1988) Sporomusa termitida sp. nov., an H&Or-utilizing acetogen isolated from termites. Arch. Microbial. 150, 282-288. Day M. F. (1938) Preliminary observations on the gaseous environment of Eutermes exitiosus Hill (Isoptera). J. Council sci. ind. Res. 11, 317-327. Liischer M. (1961) Air conditioned termite nests. Sci. Am. 205, 138-145. Matsumoto T. (1977) Respiration of fungus comb and CO, concentration in the center of mounds of some termites.
In Proceedings of the 8th International Union for the Study of Social Insects, pp. 104105. Cent. Agric. Publ. Dot., Wageningen. Messer A. C. and Lee M. J. (1989) Effect of chemical treatments on methane emission by the hindgut microbiota in the termite Zootermopsis angusticollis. Microb. Ecol. 18, 275-284. Nicolas G. and Sillans D. (1989) Immediate and latent effects of carbon dioxide on insects. Ann. Rev. Biochem 34, 97-l 16. Odelson D. A. and Breznak J. A. (1983) Volatile fatty acid production by the hindgut microbiota of xylophagous insects. Appl. Environ. Microbial. 45, 1602-1613. Rasmussen R. A. and Khalil M. A. K. (1983) Global production of methane by termites. Nature 301, 700-702. Ruelle J. E. (1964) L’architecture du nid de Macrotermes natalensis et son sens fonctionnel. In Etudes Sur les Termites Africains (Edited by Bouillon A.), pp. 327-362. Leopoldville University Press, Uopoldville. Seiler W., Conrand R. and Scharfe D. (1984) Field studies of methane emission from termite nests into the atmosphere and measurements of methane uptake by tropical soils. J. atmos. Chem. 1, 171-186. Wharton D. R. A., Wharton M. L. and Lola J. (1965) Blood volume and water content of the male American cockroach, Periplaneta americana L.-methods and the influence of age and starvation. J. Insecf Physiol. 11, 391404.
0
0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
Atmospheric carbon dioxide and acetogenesis in the termite Nasutitermes walkeri (Hill) C. M. Williams, P. C. Veivers, M. Slaytor and S. V. Cleland Department
of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia
CO, concentration inside Nasutiiermes walkeri nests is higher than in the external atmosphere. At concentrations up to 2%, CO* absorption by the termite is dependent on atmospheric CO1 concentration. Atmospheric CO, is reduced in uiuo to acetate in the termite but the amount of acetate in the termite is constant and is not related to the atmospheric CO2 concentration. Key words: Nasutitermes walkeri; Termites; Acetogenesis; Carbon dioxide
Comp. Biochem. Physiol. 107A, 113-118, 1994.
Introduction A CO* concentration higher than normal atmospheric is characteristic of nests of social insects (Nicolas and Sillans, 1989). Within the Isoptera the concentration is usually quite high and subject to both diurnal and seasonal fluctuations. For example, diurnal fluctuations in atmospheric CO2 concentration between 1.Oand 1.89% have been measured in Nasutitermes exitiosus (Day, 1938). Similarly, diurnal variations of O&2.9% (Liischer, 1961) and seasonal variations of 1.2-5.2% (Matsumoto, 1977) have been recorded within nests of the termite subfamily Macrotermitinae. Within this subfamily, Macrotermes natalensis passively controls CO2 concentration through a nest design that facilitates air circulation, but will rebuild porous parts of the nest if the internal COZ concentration increases from 1 to 2% (Ruelle, 1964). Many effects of COZ on insects have been studied including some on biochemical processes (Nicolas and Sillans, 1989). Carbon dioxide has one important role in the anaerobic metabolism of gut microbes in the paunch of termites: it acts as an electron sink for reducing power generated in the paunch (Breznak and Switzer, 1986). Reoxidation of reducing power appears to be directed towards acetogenesis rather than methanogenesis or hydrogen production as in the rumen. Thus one possible fo: M. Slaytor, Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia. Fax: 02-692-4726. Received 2 March 1993; accepted 31 March 1993. Correspondence
role for elevated atmospheric CO, in termite nests could be to ensure that acetogenesis proceeds at an optimal rate. In this paper we present data from in uivo experiments which show that atmospheric CO, can be reduced to acetate in the termite and that the amount of CO2 reduced is proportional to the atmospheric concentration but that the overall rate of acetogenesis in the termite is independent of the CO* concentration in the atmosphere.
Materials and Methods Termites Worker caste termites of Nasutitermes walkeri (Hill) collected from galleries leading to arboreal nests in suburban Sydney were used within 4 hr of collection except where otherwise indicated. Based on the weight of 100 worker caste termites, the average weight of freshly collected termites used was 8.5 mg. The paunch volume was measured by noting the increase in volume when 40 paunches were added to 0.5 ml of buffer and by measuring the decrease in weight of 10 paunches when freeze-dried, using 10 replicates. The haemolymph volume was estimated by injecting 120 nl of [‘4C]polyethylene glycol into 10 termites using the method of Wharton et al. (1965). In situ CO;! concentration in termitaria and atmospheric CO2 Air samples from within the termitaria were taken with a gas probe or, from galleries, with syringes. The arboreal nests protruded from the
113
C. M. Williams
114 tree trunk extension
between 30 and 40 cm with a vertical of 70-80 cm and were situated at
heights between 15 and 20 m. The probe was constructed from two pieces of stainless steel tubing, an outer casing (diameter 2.5 mm) enclosed a sliding insert (diameter 2.3 mm) which was secured to a needle the tip of which was covered with Parafilm’@ to prevent blockage during insertion into the nest. After the probe had been inserted to a depth indicated by markers on the outer tube, it was withdrawn slightly and the insert extended to displace the Parafilm@. Air samples were collected from the nest in 10 ml plastic syringes or from galleries in 5 ml plastic syringes. Syringes were then sealed with the needle embedded in a rubber stopper. The concentration of CO2 in 5 ml aliquots was measured by gas chromatography within 2 hr of collection using a Porapak QS column (Alltech, Deerfield, IL, U.S.A.) in a Gow Mac gas chromatograph (model 69-150). The flow rate of the helium carrier was 60 ml/min-‘, the column inlet pressure 38 kPa and the bridge current 200 mA. An Autolab Integrator (Model 23000010, Spectra Physics, CA, U.S.A.) was attached for the calculation of peak areas. Industrial grade CO2 was used as a standard. The concentration of atmospheric CO* was similarly measured.
RQ, H, and CH, estimations
Termites (3 x 100) were placed in Quickfit B14 Erlenmeyer flasks (25 ml) sealed with Subaseals and 200 ~1 or 5 ml aliquots of the atmospheric gas were removed as required for CO, analysis or O,, H, and CH, analysis, respectively. The procedure was repeated 15 times. Aliquots (5 ml) removed for 02, Hz and CH4 analysis were replaced with 5 ml of air. The aliquots were analysed at room temperature on a copper column 3 m x 6 mm) packed with a molecular sieve 5 6 60/80 (Alltech, Deerfleld, IL, U.S.A.) in a Hewlett Packard gas chromatograph (model 5710) with a thermal conductivity detector. The following conditions were used: carrier gas, argon; flow rate, 20 ml/min-‘; column temperature, 150°C; detector temperature, 100°C; sample loop, 5 ml. The chromatograph was linked to a Hewlett Packard integrator and the column was calibrated with appropriate standards. 200 ~1 aliquots removed for COZ measurement for RQ estimations were analysed at room temperature on a Porapak QS (Alltech) column in a Gow Mac (model 69-150) gas chromatograph fitted with a thermal conductivity device. Bridge current, 200 mA; carrier gas, helium, flow rate, 60 ml/min-‘. The
et al.
concentration of CO* was calculated from peak heights and was linear over the range used (up to 10%). O2 utilization under non -atmospheric concen trations of CO2 One hundred worker caste termites were placed in Quickfit B14 Erlenmeyer flasks (25 ml) which were then fitted with Subaseals. COZ atmospheres were established with industrial grade CO*. Gas samples (200 ~1) were taken, over a 5 hr incubation at 25°C and analysed for O2 by injecting into a copper column (3 m x 6 mm) packed with a molecular sieve 5 8, 60/80 (Alltech) in a Gow Mac gas chromatograph (model 69-150). The flow rate of the helium carrier was 60ml/min-‘, the column inlet pressure 38 kPa and the bridge current 200 mA. An Autolab Integrator (Model 23000010, Spectra Physics, CA, U.S.A.) was attached for the calculation of peak areas. Ambient air (20.946% 0,) was used as a standard. 14C02 absorption from atmospheric
and [14C]acetate 14C02
production
was prepared from NaH14C03 ‘TO2 (0.1 mCi/mmol; Amersham International, U.K.) or Ba14C03 (51 mCi/mmol; Amersham). Different partial pressures of CO* were established in stoppered 25 ml Erylenmyer flasks containing 50 termites by injection of 14C02. Termites were either incubated for 1 hr at 25°C under varying 14C02 atmospheres or were incubated at 25°C for varying periods of time at constant CO* (2%). After incubation, termites were probe-sonicated (4°C for 30 set at 40 W; Branson model B 12 sonicator) in 2 M NaOH (600 ,ul) and then centrifuged (15 OOOg for 10 min at 4°C); aliquots were used to determine the total amount of 14C-labelled material in the termite. Aliquots (200 ~1) were placed in the outer well of a Warburg flask (30 ml) and the 14C02 liberated with 500~1 2 M HCI was trapped over 4 hr with 300 ~1 of freshly prepared 2 M NaOH in the centre well. Aliquots (100 or 200 ~1) from the outer well were used for HPLC analysis, enzymatic determination of CH,COOH (Boehringer Mannheim kit no. 148 261) and scintillation counting to determine incorporated 14C02. Aliquots from the inner well were used to estimate “C02. Aliquots (100 ,ul) for scintillation counting were applied to glass microfibre filters (2.1 cm; Whatman International Ltd, U.K.) impregnated with 100 ~1 of freshly prepared 10% Ba(CH,COO), . Dried filters were placed in 20 ml glass scintillation vials containing 5 ml of scintillation fluid (0.6% PPO in toluene).
CO2 and acetogenesis in termite
HPLC The acidified homogenate from the outer well was filtered through a 0.45 pm Millipore filter and injected through a Rheodyne 7125 syringe loading sample injector using 25-200 ,ul syringes (SGE, Melbourne) into an HPLC apparatus which consisted of an LKB Liquid Chromatograph containing the following modules: a Model 2152 HPLC controller, a 2150 pump and a 2040-203 gradient mixing valve. Absorbance was monitored at 200 nm with a 2151 variable wavelength monitor and data were recorded and integrated by the Maxima 820 Chromatography work station (Dynamic Solutions). A Bio-Rad Aminex HPX-87H Organic Acid Analysis (cation-exchange) column (300 x 7.8 mm), fitted with a 5 cm H+ guard column (Bio-Rad 1250129; 40 x 4.6 mm) containing the same resin was used. Samples were eluted isocratically with 5 mM HzS04 (flow rate 0.5 ml/mine’) at 45°C. Fractions eluting from the column were collected, on the basis of retention time, dropwise into scintillation vials, using a Gilson Fraction Collector (Model 202). The metabolites were identified by comparison with retention times of standards. The distribution of 14C0, in metabolites was determined by the level of radioactivity in collected fractions. The pH was adjusted to 8-9 by addition of 2.5 M KOH and 0.63 M KHC03 (10-100 ,ul depending on fraction volume). All fractions were dried overnight at 7o”C, redissolved in 100 ,~l of milli-Q water and 5 ml of scintillation fluid (Optiphase “HiSafe” 3; LKB Scintillation Products) added. Scintillation counting Radioactivity was measured either in a Beckman (model LS 3800) or in a Wallac (model 1410; Pharmacia) liquid scintillation counter. Samples processed by the Beckman counter were corrected for quenching using NaH14C0, as a standard; the Pharmacia counter was programmed to correct for quenching and chemoluminesence automatically. Standards for 14C0, were prepared by absorbing aliquots of the experimental gas mixture in Soluene@.
115
at depths of 10 and 15 cm analysed for mean values of 0.48 f 0.08% (n = 2) and 0.68 + 0.03% CO, (n = 2), respectively. It was not possible to probe beyond 20cm without bending the probe. The fragility of the galleries made gas sampling difficult but mean values of 0.14 + 0.03% (n = 4) were recorded from intact galleries from two different nests on three separate days. Same day samples (n = 2) from one nest indicated mean concentrations of 0.14 * 0.01% co,. When freshly collected worker caste termites of N. walkeri were incubated in air at 25°C they consumed O2 and produced COZ at 82 + 2 and 87 It 2 nmol termite-’ hr-’ respectively. Hydrogen was produced at 0.74 + 0.05 nmol termite-’ hr-‘. The amount of CH4 produced (N 1 nmol termite-’ hr-’ ) was on the lower limit of detection. The RQ value for all determinations was in the range 1.1 + 0.14. O2 utilization slowly decreased at higher CO, concentrations from a rate of 83 + 5 nmol O2 termite-’ hr-’ (n = 6) at normal atmospheric CO2 concentration though, at atmospheres of 2% COZ the utilization was not significantly altered (80 k 13 nmol O2 termite-’ hr-’ (n = 2) (Fig. 1). At CO2 concentrations higher than shown in Fig. 1, the rate of 0, utilization decreased dramatically. It declined to 41 k 7 (n = 3) and 12 & 6 (n = 2) nmol termite-’ hr-’ at CO, concentrations of 75 and 90%, respectively. Termites appeared to be normally active during incubation at concentrations of CO2 less than or equal to 5% but ceased moving within 1 min of being exposed to CO* concentrations of 75% and greater. Incubating termites in 14C02 atmospheres containing increasing amounts of total CO2 showed that the amount of “C-labelled material found in the termite was linear between 0.5 and 2% atmospheric CO2 and that the rate of uptake was 26 nmol termite-’ hr-’ %-’ CO*. At higher concentrations (up to 5%) the rate decreased (Fig. 2). Thus, 2% CO2 appeared to be the CO2
Statistical analysis All errors are represented by the standard error of the mean. Correlation coefficients for calculated rates are expressed as r.
Rl?SUltS
501
’ 0
Nasutitermes walkeri is common in the Sydney region and the galleries on the bark connecting the nest with the ground allow for multiple and rapid collection of workers. Gas samples for CO, estimation, collected from two nests,
1
2
3
4
5
Atmospheric CO2 (%) Fig. 1. Oxygen utilization as a function of atmospheric CO2 concentration in N. walkeri. The 0 sample is at normal atmospheric CO, concentration (0.03%).
C. M. Williams et al.
0
0
1
2
3
4
5
Atmospheric Ct& (%)
20
60
40 Time (min)
Fig. 4. Uptake of CO, by N. w&eri as from an atmosphere containing 2% CO,.
Fig. 2. Uptake of CO, by N. wnlkeri as a function of atmospheric CO, concentration.
over 60min. In two experiments, the rate of [14C]acetate production over 1 hr from absorbed concentration at which absorption of 14C02 is 14C02 at 2% atmospheric CO2 was 1.77 optimal and was used for most incubations. (r = 0.99) and 2.80 (r = 0.98) nmol terWhen termites were incubated at 0.7% CO*, mite-’ hr-‘; this corresponds to rates of CO* 60% of the absorbed radioactivity was in the incorporation of 3.5 and 5.6 nmol CO2 tertermite body, the remainder being in the gut mite-’ hr-‘. The incorporation of 14C02 into (Fig. 3). At least 91% of the “C-labelled ma- [14C]acetate over the first 10min in the first of terial was 14C02. At 2% the absorption of 14C02 these experiments, when the rate of incorporwas linear over the first 30 min (rate 0.64 nmol ation is rapidly changing, is shown in Fig. 5. 14C02 termite-’ min-’ ) but appeared to de- Acetate was the main metabolite formed from crease over the next 30 min (0.23 nmol 14C0, 14C0, on the basis of HPLC data. Two minor termite-‘min-‘) (Fig. 4). The experiment was metabolites were not identified: formate, propirepeated: the trends were similar, although the onate, butyrate, lactate and pyruvate were elimrate of 14C0, absorption in the second experinated as possibilities. The total amount of iment was approximately twice that of the first. acetate in whole termites in these experiments At least 9% of the absorbed 14C02 was incorwas 13.9 kO.4 (n = 8) and 19.3 kO.2 (n = 6) porated into metabolites, corresponding to rates nmol acetate/termite. The rate of acetogenesis of COz incorporation of 3.2 and 6.4 nmol ter- as a function of atmospheric CO, did not appear mite-’ hr-‘, respectively, for the two experto increase beyond atmospheres of greater than iments. The rate of 14C02 incorporation into 3% (Fig. 6). metabolites at 2% atmospheric CO* was linear The paunch of N. walkeri has a volume of 1.5 + 0.2 ~1 (n = 4) using the volumetric method and 1.49 f 0.08 ~1 (n = 10) using the freeze drying method, and contains 12 + 1 nmol of acetate (n = 15); the concentration is thus approximately 8 mM. The haemolymph volume was g f 1.5 + 0.1 ,ul (n = 10) and the acetate concentration was 5 mM. 2 sg g 20-
3o
-
lo0
0
1
2
3
Time (h)
Fig. 3. Uptake of CO, into the gut and body of N. walked. l gut; 0 body. Termites were incubated for 3 hr in the presence of 0.7% CO, prepared as described in the text. Samples were taken at the times indicated. Ten termites were degutted and the guts and bodies mashed and placed overnight in scintillation vials at 50°C containing Soluene& (1 ml). After cooling, scintillation fluid was added and radioactivity estimated in a Beckman (mode1 Is 3800) scintillation counter. Samples were corrected for quenching using [U-‘4C]glucose as standard.
0
2
4
6
8
10
Time (min) Fig. 5. Acetopnesis in N. walkeri from atmospheric CO,; concentration of COr in atmosphere, 2%.
CO, and acetogenesis in termite
117
Cubitermes speciosus acetogenesis is a minor process compared to methanogenesis (Brauman et al., 1990). Earlier gloomy predictions of global catastrophe due to overproduction of CH, by termite activity have fortunately not occurred (Rasmussen and Khalil, 1983) but methanogenesis is clearly an important process in many termite genera (Seiler et al., 1984). Our in vivo results clearly show that atmospheric CO2 can be reduced to acetate. As the overall 0 1 2 3 4 5 rate of acetogenesis does not increase with increasing CO2 concentrations, the atmospheric Atmospheric CO,(%) CO, presumably exchanges with that in the Fig. 6. Rate of acetate formation in N. walkeri from body and the paunch. This exchange is quite atmospheric CO, as a function of atmospheric CO,. rapid as acetate derived from atmospheric CO* can be detected within 2 min and that within 10 min the rate of production is 50% that of the maximum rate. Discussion On the calculation that glucose is the only The concentration of CO2 in both the nest carbohydrate being assimilated, a reasonable and the galleries of Nasutitermes walkeri is assumption as 85% of carbohydrate metabsignificantly higher than that of air though the olised in R. flavipes is glucose (Odelson and Breznak, 1983), then the rate of 0, utilization concentration in the nest is lower than that reported for N. exitiosus (Day, 1938) and other indicates that glucose is being metabolized in N. mound builders. Nest COr measurements on walkeri at about 14 nmol termite-’ hr-‘. In R. N. walkeri were made on early morning j7avipes, one-third of acetate production is as a samples, known to be the lowest diurnal level result of CO2 reduction (Breznak and Switzer, 1986). Hence it is reasonable to suppose that in in N. exitiosus (Day, 1938). Nasutitermes N. walkeri, acetogenesis from CO, reduction is walkeri is the first arboreal nest building termite on which CO* measurements have been proceeding at 14 nmol termite-’ hr-‘. Our results show that about 10% of this can come recorded. The increasing CO, gradient towards the centre of the nest in N. walkeri was also in vivo from atmospheric CO, when the atmosfound in N. exitiosus (Day, 1938) and may be pheric CO* concentration is 2%. On the basis of O2 utilization, the overall rate related to the distribution of termites within the of metabolism is hardly affected by increasing nest. atmospheric CO, concentration which also does The RQ values for freshly collected termites, though variable over a comparatively wide not affect the total amount of acetate in the range, appear centred on 1.0 indicating that termite. A maximum rate of acetogenesis of only carbohydrate, presumably cellulose, and 0.6 nmol termite-’ hr-’ from atmospheric COz not lignin is being digested (Odelson and Brez- occurs in atmospheres of about 3% CO*, As nak, 1983). The moderate decline in 0, utilis- this rate is maintained, but not exceeded, at ation over the physiological CO, atmospheric higher CO, atmospheres it presumably repconcentrations tested is consistent with the well- resents a steady state. In vitro experiments with documented anaesthetic effects of CO2 in insects gut homogenates of R. flavipes indicate that (Nicolas and Sillans, 1989). The small amounts Hz can be used for acetogenesis and that acetoof H, and CH, evolved suggest that most of the genesis is dependent on Hz concentration reducing power produced in the anaerobic (Breznak and Switzer, 1986). A H&O,-utilizpaunch is used for acetogenesis from CO* as it ing acetogen has been isolated from the gut of is in other nasutes such as N. costalis and N. N. nigriceps (Breznak et al., 1988). These expernigriceps (Breznak and Switzer, 1986). On a iments, however, do not show that H, is the molar basis, the evolution of H, or CH, was in vivo reducing agent. The absence of for-mate about 1% that of O2 consumption or CO2 or propionate as in vivo reduction products of production as has also been reported in the atmospheric CO2 in N. walkeri is in contrast to lower termite Reticulitermes flavipes (Odelson in vitro results obtained from gut homogenates and Breznak, 1983). Not all termites are as of N. costalis and N. nigriceps (Breznak and economical with the available reducing power Switzer, 1986). from fermentation and squander it in CHI proOur major conclusion is that although duction. For example, Zootermoposis angustiatmospheric CO2 can be reduced to acetate collis produces 10.3 nmol termite-’ hr-’ of CH, in vivo in N. walkeri, this process is not (Messer and Lee, 1989) and in the soil-feeder needed to achieve a maximum rate of
118
C. M. Williams et al.
acetogenesis and thus a biochemical explanation for the high CO, concentration in the nest remains unanswered. Acknowledgemenrs-This work was supported in part by a grant from the Australian Research Council.
References Brauman A., Labat M. and Garcia J. L. (1990) Preliminary studies on the gut microbiota of the soil feeding termite: Cubitermes speciosus. In Microbiology in Poecilotherms (Edited by L&e-l R.), pp. 73-77. Elsevier, Amsterdam. Breznak J. A. and Swit& J. M. (1986) Acetate synthesis from H, plus CO, by termite gut microbes. Appl. environ. Microbial. 52, 623630.
Breznak J. A., Switzer J. M. and Seitz H.-J. (1988) Sporomusa termitida sp. nov., an H&Or-utilizing acetogen isolated from termites. Arch. Microbial. 150, 282-288. Day M. F. (1938) Preliminary observations on the gaseous environment of Eutermes exitiosus Hill (Isoptera). J. Council sci. ind. Res. 11, 317-327. Liischer M. (1961) Air conditioned termite nests. Sci. Am. 205, 138-145. Matsumoto T. (1977) Respiration of fungus comb and CO, concentration in the center of mounds of some termites.
In Proceedings of the 8th International Union for the Study of Social Insects, pp. 104105. Cent. Agric. Publ. Dot., Wageningen. Messer A. C. and Lee M. J. (1989) Effect of chemical treatments on methane emission by the hindgut microbiota in the termite Zootermopsis angusticollis. Microb. Ecol. 18, 275-284. Nicolas G. and Sillans D. (1989) Immediate and latent effects of carbon dioxide on insects. Ann. Rev. Biochem 34, 97-l 16. Odelson D. A. and Breznak J. A. (1983) Volatile fatty acid production by the hindgut microbiota of xylophagous insects. Appl. Environ. Microbial. 45, 1602-1613. Rasmussen R. A. and Khalil M. A. K. (1983) Global production of methane by termites. Nature 301, 700-702. Ruelle J. E. (1964) L’architecture du nid de Macrotermes natalensis et son sens fonctionnel. In Etudes Sur les Termites Africains (Edited by Bouillon A.), pp. 327-362. Leopoldville University Press, Uopoldville. Seiler W., Conrand R. and Scharfe D. (1984) Field studies of methane emission from termite nests into the atmosphere and measurements of methane uptake by tropical soils. J. atmos. Chem. 1, 171-186. Wharton D. R. A., Wharton M. L. and Lola J. (1965) Blood volume and water content of the male American cockroach, Periplaneta americana L.-methods and the influence of age and starvation. J. Insecf Physiol. 11, 391404.