On the respiratory quotient (RQ) of termites (Insecta: Isoptera)

Pergamon PII: S0022-1910(97)00036-X

J. Insect Physiol. Vol. 43, No. 8, pp. 749–758, 1997  1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0022-1910/97 $17.00 + 0.00

On the Respiratory Quotient (RQ) of Termites (Insecta: Isoptera) L. NUNES ,* D. E. BIGNELL,† N. LO,‡ P. EGGLETON§ Received 21 November 1996; revised 18 February 1997

The respiratory quotient (RQ) at 28°C was determined by Warburg manometry in 23 species of termites from the Mbalmayo Forest Reserve (Cameroon) and three sub-tropical species cultured under laboratory conditions in the U.K. or freshly collected in Australia. The data are tabulated with other recently reported RQs (determined by manometry or GC) and with measured CH4 emission rates to provide a survey of 29 species covering both lower and higher termites in all major trophic (functional) categories. In all species, except the wood-feeding Coptotermes acinaciformis and the soil-feeding Cubitermes fungifaber, the observed mean values (with manometry corrected for known fluxes of H2 and CH4) were at or well above 1.00. Soil-feeding forms (except C. fungifaber) generally showed a high apparent RQ (not corrected for H2), with nine species (out of 13) above 1.20 and six species above 1.30. Wellreplicated laboratory experiments with Reticulitermes lucifugus showed that there was a tendency for RQ to fall with time over a 4-h incubation, although remaining greater than 1.00. The observed RQs are consistent with carbohydrate being the principal substrate supporting respiration in all trophic and taxonomic categories, with little or no contribution from the degradation of lignin or other polyaromatic materials. However, in many species (especially soil-feeders), the observed RQ is greater than that expected from known fluxes of O2, CO2 and CH4 on the assumption that carbohydrate is the respiratory substrate. This presupposes that there is a large hydrogen sink (additional to CH4 production), possibly the emission of H2 gas, and/or the existence of unresolved digestive mechanisms or electron routings. Uncertainties in the use of manometry with termites are discussed.  1997 Elsevier Science Ltd Termite Respiration Hydrogen emission Methane emission Carbohydrate degradation

INTRODUCTION

of about 0.8 (e.g. Cantarow and Schepartz, 1968; Guyton, Respiratory quotient (RQ) is a classical concept of ani- 1971; Hoar, 1975; Stanier and Forsling, 1990). Values mal physiology, defined as the molar ratio of carbon outside this range (i.e. 0.7–1.0) are relatively rarely dioxide formed and oxygen consumed per unit time dur- encountered, although RQs below 0.7 can be associated ing the metabolism of foodstuffs (Richardson, 1929; with gluconeogenesis, and transient increases above 1.0 Schmidt-Nielsen, 1975). Tissues or whole organisms oxi- can result from the conversion of carbohydrate into fat dizing pure carbohydrate show an RQ of 1.0, those oxid- (Cantarow and Schepartz, 1968). In mammals, few izing fat an RQ of 0.7 and those oxidizing protein an RQ reliable reports place RQ above 1.10 under non-pathological conditions (Guyton, 1971), but values as high as 1.49 were found in force-fed geese (Benedict and Lee, *Department of Biology, Imperial College, University of London, Lon- 1937). don SW7 2BB, U.K. In insects, similar investigations have focussed on the †School of Biological Sciences, Queen Mary and Westfield College, identification of substrates used during flight or during University of London, Mile End Road, London E1 4NS, U.K. embryonic development [reviews by Gilmour (1965), ‡Department of Biochemistry, The University of Sydney, Sydney, NSW 2006, Australia Bailey (1975), Beenakkers et al. (1981) and Steele §Biodiversity Division, Entomology Department, The Natural History (1981)]. The range of values observed is generally simiMuseum, Cromwell Road, London SW7 5BD, U.K. lar to that of vertebrates, although the conversion of glu储To whom all correspondence should be addressed. Fax: 0181 983 cose to lipid is again reported to elevate RQ above 1.00, 0973; E-mail: [email protected] Present address: Nucleo de Madeiras, Laboratorio Nacional de and transient values as high as 2.00 have been noted (e.g. Engenharia Civil, Av. do Brasil 101, 1799 Lisboa Codex, Portugal. Zebe, 1953). 749

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A number of determinations of RQ in termites have been reported, mostly early studies designed to define the digestive contribution of the gut microbiota by comparing gas exchanges in normal and defaunated insects [reviews by Peakin and Josens (1978) and Grasse´ (1982)]. Lower termites (those having intestinal protozoans) were reported by Cleveland (1925), Cook (1932), Hungate (1938), Gilmour (1940), Ghidini (1939) and Lu¨scher (1955) to have values close to unity in the worker caste (at temperatures ranging 19–25°C). Starvation or treatments that eliminated the gut flagellates reduced RQ to about 0.8, which is consistent with the normal digestive process being a symbiont-assisted degradation of polysaccharide, yielding carbohydrate substrates for metabolism. Larvae, alates and soldiers (which lack the gut microbiota or are fed by workers) generally had RQs in the range 0.78–0.93. Cook (1932) and Gilmour (1940), working with Zootermopsis nevandensis, noted that CH4 and H2 were produced by normal workers, in addition to CO2, and corrected their calculations of RQ accordingly. In this species, CH4 and H2 evolution was equivalent to about 10% of the rate of O2 uptake, and the corrected RQ (assuming complete carbohydrate dissimilation) is 1.05, close to the apparent value of 1.09 actually observed (Gilmour, 1940; Peakin and Josens, 1978). Information on RQ in higher termites (where the gut microbiota is exclusively prokaryotic) is fragmentary. Day (1938) reported values of 0.89 and 0.85 for workers of Eutermes ( = Nasutitermes) exitiosus incubated at 27°C, whereas in workers of Nasutitermes costalis, the RQ at 21–25°C was 0.90 (Wiegert and Coleman, 1970). He´brant (1970) examined the soil-feeding Cubitermes exiguus, and determined the RQ to be 0.9 at 30°C, declining to 0.7 for workers examined in isolation from their mounds, but the gas exchanges of intact colonies suggested a value of 1.0. Rouland et al. (1993) measured RQ in a suite of 15 species of higher termites from equatorial Africa, including xylophagous, litter-feeding and soilfeeding forms. The values obtained (at 29°C) ranged from 0.36 to 0.98, but were generally lowest in the soil-feeders. The observation of RQs below, or well below 1.00 in higher termites is paradoxical. Many species produce quite large amounts of CH4 and H2 (especially soilfeeders); furthermore, there is as yet no convincing evidence that the basic digestive processes differ from those of lower termites (Slaytor, 1992; Bignell, 1994). However, the precise identity of substances degraded by soilfeeding forms and other termites feeding on highly decayed organic matter is not known, and it has been suggested that lignin-derived polyaromatic compounds may be of importance (Rouland et al., 1993; Bignell, 1994; Brune et al., 1995b). In such circumstances, the RQ should be less than 1.00 as, in comparison with carbohydrates, the oxidation of aromatic polymers to CO2 requires more O2 per C atom. In this paper, we report determinations of RQ (by Warburg manometry)

and of CH4 production (by GC) in 26 species of lower and higher termites made under either laboratory or field conditions. The results show that almost all RQs (corrected for H2 and CH4) and apparent RQs (corrected for CH4 only) are above 1.00, supporting the case that carbohydrates are the principal substrates degraded in all trophic groups. To minimize confusion, in this paper, the term RQ will be used with data on CO2 production and O2 consumption, where corrections have been made for both H2 and CH4 effluxes. In other instances, where data on CH4 efflux alone are available, the estimation of O2 consumption rates by manometry may be in error owing to the possible production of an unknown quantity of H2. In these cases, the quotient of O2 consumed to CO2 produced will be referred to as ‘apparent RQ’ (RQapp). In references to existing published estimates of RQ, the term is used in the sense employed by the authors concerned, whether or not corrections for H2 and CH4 were made to manometric data. MATERIALS AND METHODS

Laboratory termites Reticulitermes lucifugus was collected In the Reserva Natural da Arrabida, Portugal, air-freighted to the U.K. and maintained at a constant temperature (27 ± 1°C) and high relative humidity (⬎75%) on a diet of filter paper and Scots or maritime pine wood. R. santonensis was obtained from the Building Research Establishment (Watford, U.K.) and maintained under the same conditions. Small colonies of Macrotermes subhyalinus were reared in the laboratory as described by Anklin-Mu¨hlemann et al. (1995). Field-Sampled termites Twenty-two species of higher termites (Isoptera: Termitidae) and a single species of lower termite, Schedorhinotermes putorius (Isoptera: Rhinotermitinae) were freshly sampled from old plantation and mature secondary forest in the Mbalmayo Forest Reserve, southern Cameroon. Manometers were set up in the Field Station of International Institute of Tropical Agriculture, within the Reserve. Termites (worker caste) were sorted on moistened filter paper to remove damaged or moribund individuals before being placed in the flasks. A 30-min equlibration period was allowed before manometers were closed (see below). Generally, no more than 1h elapsed between sampling and the start of incubation. Termite species chosen for physiological studies were representative of the major trophic groups found in the forest assemblage and, wherever possible, those that contributed most to overall biomass (Eggleton et al., 1996). However, some relatively rare species that are conspicuous and can be sampled in large numbers (Macrotermes mulleri, Cephalotermes rectangularis and Termes hospes), were also included. Coptotermes acinaciformis

RESPIRATORY QUOTIENT OF TERMITES

(a lower termite) was freshly collected from infested Pinus radiata in the Pennant Hills State Forest Reserve, NSW, Australia. Manometry Constant-volume Warburg manometry was chosen for the determination of RQ. The advantage of this method, in addition to its simplicity, accuracy and reliability, is that under field conditions, power is required only to maintain a water bath at constant temperature. After sampling, termites were enclosed in single side-arm Warburg manometer flasks, 13–16 ml internal volume (approx. 150 mg fresh weight biomass per flask for most experiments, except where RQ was determined as a function of the weight of termites per flask). For each determination, six flasks were employed in two arrays of three flasks on either side of a rectangular perspex water bath of 8 l capacity, maintained at 28°C and stirred with a Bioblock Thermostatic heater (Jeulin, Evreaux, France). Each array comprised a thermobarometer (containing 1 ml H2O), an experimental flask (containing termites and 0.1 ml 1 M KOH in the centre well) and a control flask containing termites only. In a few cases where termite numbers were limited, a single array only was employed. Flasks and manometers were paired, standardized gravimetrically using mercury and water, and tested for gas-tightness by the hydrazine/ferricyanide method of Umbreit et al. (1964). Kreb’s fluid was employed in the manometers, which were adjusted to constant volume at 30-min intervals during an incubation of 3h. xO2 was calculated with adjustment of the raw volumetric data for CH4 production (determined separately, see below): a volume (␮l) estimated as the CH4 produced by the fresh weight of termites placed in the flask was added to each manometer reading for the KOH flask (U-tube scales were graduated in ␮l). No adjustment was made to data from the control flask, but the calculation of xCO2 utilized the corrected O2 consumption (Cook, 1932; Umbreit et al., 1964). Additional corrections for H2 were made in the same way where efflux data were available. Termite mortality was determined at the completion of incubation; where this exceeded more than one individual per flask, results were discarded. Replicate sampling and incubations were made according to the availability of termites. Manometry with R. lucifugus and R. santonensis was carried out by a method essentially similar to that described above. The variants were: water bath temperature, 27.0 ± 0.5°C; equilibration, 60 min; duration of incubation, 240 min; absorption of CO2, 20% KOH. Flask constants were calculated from data supplied by the manufacturers. For Coptotermes acinaciformis, manometry was carried out at 25°C by a similar method. CH4 and H2 effluxes Field-sampled termites (approx 150 mg) were sorted on moistened filter paper to remove damaged individuals and weighed before being placed in a 100-ml amber glass

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gas sampling bottle (Chromatography Services), sealed by a Chrompack aluminium crimp cap (20-mm diameter) fitted with a Teflon-lined rubber washer. Twenty millilitres of headspace gas were withdrawn with a hypodermic syringe at 1, 2 and 3h intervals and transferred to similarly sealed 50-ml Chrompack glass gas sampling vials from which 20 ml of air had previously been withdrawn by syringe. Methane field standards were established at the same time by injecting 20 ml of a 100 ppm mixture of CH4 in N2 (Phase Separations) into identical vials. Gas sampling vials were analysed for CH4 in the U.K. by the method of Anklin-Mu¨hlemann et al. (1995). Termite mortality was determined at the conclusion of the incubation, and the results from groups in which more than one individual had died during incubation were discarded. For single mortalities, an adjustment to calculated effluxes was made, assuming no fluxes occurred from the individual concerned. Methane effluxes were calculated for each hour of the 3-h of incubation and averaged. Replicate incubations were made, according to the availability of termites; where n⬎2, CH4 efflux is given as mean ± SD. RQ and CH4 effluxes were determined from the same batch of termites. R. lucifugus and R. santonensis were incubated for 4h at 25°C in 25-ml cylindrical vials containing a sliver of dry filter paper and closed with screw caps fitted with silicone rubber seals. The headspace gases were sampled and analysed for CH4 as described by Anklin-Mu¨hlemann et al. (1995). For H2, 0.5-ml aliquots of headspace gas were injected into Shimadzu model 14-A gas chromatograph fitted with a Chromosorb 102 S/S column, using argon as the carrier gas at 20 ml min−1. The column was maintained at 30°C and H2 detected by thermal conductivity at 110°C. A standard of 94 ppm in air (20.8% O2, balance N2; Gas Measurement Instruments) was employed. The measurement of CH4 and H2 effluxes from M. subhyalinus is described by Anklin-Mu¨hlemann et al. (1995). In these laboratory-maintained termites, RQ and the effluxes of CH4 and H2 were generally determined in separate batches from the same colony. Derivation of RQ and RQapp RQ was computed by the direct method set out in Umbreit et al. (1964). As termites in the KOH flask respire in the absence of CO2, this method assumes that CO2 concentration has no effect on the rate of respiration. KOH flasks and control flasks (containing termites only) were paired randomly for the calculation. For most incubations, there were, therefore, two pairings between KOH and control flasks. In each pairing, RQ was calculated for each of five 30-min periods corresponding to the last 150 min (or the last 210 min where appropriate) of the incubation and averaged to provide an overall value. Gas fluxes during the first 30 min of incubation, which show greater variability, were not included in the calculation.

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Derivation of theoretical O2 consumptions Where CH4 (and, in some cases, also H2) effluxes are known, it is possible to calculate a theoretical O2 consumption based on the assumption that carbohydrate is the substrate being metabolized, but is incompletely oxidized, yielding CO2, CH4 and H2 in the quantities (relative molar ratios) indicated by the measured effluxes. This is done as follows: Step 1: moles of CO2 and CH4 are summed to give the number of moles of (CH2O) consumed ( = nCH2O). Step 2: the number of H in the H2 and CH4 produced is subtracted from the H in nCH2O and divided by 2. This gives the number of moles of H2O produced. Step 3: the number of O in the CO2 and H2O produced is summed. The number of O in nCH2O is then subtracted from this total and divided by 2. This gives the number of moles of O2 needed to metabolize nCH2O, and produces a balanced empirical equation for termite respiration.

RESULTS

Survey of RQ, RQapp and the effects of trophic habit Table 1 shows measured RQ values (RQapp where manometric data were corrected only for CH4 eflux) and available information on other gas exchanges (CH4, H2, N2), for 29 species of termites in 24 genera. Both lower and higher termites are included, and in the latter category, there are representatives of all four inclusive subfamilies; Macrotermitinae, Apicotermitinae, Nasutitermitinae, Termitinae. Group I contains the three species for which complete gas exchange data are available (Coptotermes acinaciformis, Nasutitermes walkeri, Reticulitermes flavipes). Group II incorporates three species for which both CH4 and H2 fluxes are available (but not rates of N2 fixation), whereas the remaining 23 species, for which only CH4 fluxes have been determined, constitute Group III, subdivided by trophic habit (sensu Eggleton et al., 1995). In all but two cases (Coptotermes acinaciformis and Cubitermes fungifaber), mean RQ or mean RQapp was greater than 1.00, ranging up to 1.65 ± 0.18 in the inquilinous soil-feeding species Ophiotermes grandilabius. Other species were less than 1.40, although variance was great in some cases. In some determinations, flasks can run below 1.00 throughout the incubation or drop below 1.00 in the later stages. The highest RQapp values were found amongst the soil-feeding species (arithmetical mean of 13 species, 1.26), but this trophic group also contained two species (Cubitermes fungifaber, Pericapritermes amplignathus) with lower values (0.94 ± 0.19 and 1.04 ± 0.14, respectively). C. fungifaber was the only soil-feeding species examined to show a mean RQapp ⬍ 1.00, although its physiology and ecology are not obviously different from other humivorous forest species.

Table 1 permits an examination of the relationship between RQ (or RQapp) and methane production. In species with low CH4 effluxes (generally wood- or litter-feeding forms producing ⬍ 0.100 ␮mol g−1 h−1), RQ or RQapp is also towards the lower end of the range observed in this survey ( ⬍ 1.12). Conversely, the soil-feeders producing methane in the range 0.147–0.342 ␮mol g−1 h−1 have higher RQs, although C. fungifaber (methane 0.263 ␮mol g−1 h−1; RQapp = 0.94), P. amplignathus (methane 0.319 ␮mol g−1 h−1; RQapp = 1.04) and T. macrothorax (methane 0.242 ␮mol g−1 h−1; RQapp = 1.09) are exceptions. Termes hospes, in which the methane efflux is an exceptional 0.798 ␮mol g−1 h−1, has an RQapp of 1.33. Effect of time of incubation and termite crowding on RQ To determine whether the conditions of incubation could affect the results, RQs were determined for R. lucifugus at 30-min intervals for a total of 4h, in an experiment with a larger replication (13 determinations). Corrections to manometric data were made for CH4 and H2 effluxes. Fig. 1 shows that there is a decline of mean RQ with increased time of incubation. Non-parametric ANOVA by the Kruskal–Wallis test (Sokal and Rohlf, 1969) showed that there was a significant variation with time of incubation (P ⬍ 0.001). A posteriori pairwise comparisons by the Mann–Whitney U-test showed that incubation for 60 min produced a significantly greater RQ and, for 210 or 240 min, a significantly lower RQ than incubations of intermediate duration (P ⬍ 0.01). RQs of termites incubated for 120, 150 and 180 min were not significantly different. In most cases, the observed RQs were well above 1.00, inferring that carbohydrate was the substrate utilized to support respiration. The decline of RQ with time may indicate that the insects move towards a starvation metabolism as the time of incubation increases without the opportunity to feed. Higher termites freshly sampled in the field usually showed a trend for RQapp to be reduced with time of incubation (data not shown). In most cases, the magnititude of this change was about 0.15–0.20 units of RQ for the last 150 min of incubation (i.e. the first 30 min excluded). Therefore, whereas some termites show a tendency for RQ to diminish with time of incubation, this does not detract from the general conclusion that, in most termites, RQ or RQapp has values above 1.00. A smaller data set showing the effects of the degree of crowding of R. lucifugus in the flask on RQ (measured over 4h) is shown in Fig. 2. Flask volumes were 13– 16 ml, as in all other manometry. A regression line can be drawn, suggesting a trend towards reduced RQ at higher termite density, but there is not a significant correlation (r2 = 0.361, P⬎0.05). A pilot investigation of the effects of flask crowding in higher termites (A. quietus, C. heghi, T. hospes and T. macrothorax; 20–100 individuals per flask, data not tabulated) failed to show any effect of crowding (P⬎0.05), but small numbers of individuals ( ⬍ 20 for small species under 5 mg, ⬍ 10 for

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TABLE 1. Known fluxes of oxygen, methane and hydrogen gases, known rates of nitrogen fixation and observed respiratory quotients (RQ or RQapp) in 29 species of termites Termite

Group I (data for N2 fixation available3) Coptotermes acinaciformis Nasutitermes walkeri

4 5

O2 consumption ␮mol g−1 h−1 mean ± SD1

CH4 efflux ␮mol g−1 h−1 mean ± SD1

H2 efflux ␮mol g−1 h−1 mean ± SD1

RQ or RQapp (corrected)2 mean ± SD

13.66 ± 0.97 9.947 ± 0.941 (n = 15) 15.90 ± 1.93

0.310 ± 0.040

0.210 ± 0.030

0.94 ± 0.14 (n = 8)

0.210 ± 0.050

0.087 ± 0.005

1.05 ± 0.01 (n = 5)

0.134 ± 0.045

0.134–0.268

1.03

± ± ± ±

1.00 ± 0.01 (n = 4) n.d. 1.16 ± 0.10 (n = 18) 1.16 ± 0.16 (n = 15)

Reticulitermes flavipes Group II (data for N2 fixation unavailable) 13.772 (n = 2) Macrotermes subhyalinus MAJ6 n.d. Macrotermes subhyalinus MIN6 Reticulitermes lucifugus 17.42 ± 1.72 Reticulitermes santonensis 18.30 ± 2.39 Group III (data for N2 fixation and H2 efflux unavailable) (Litter feeders) Macrotermes mulleri MAJ 11.300 (n = 2) Macrotermes mulleri MIN 10.090 (n = 2) Pseudacanthotermes militaris 11.940 (n = 2) (Wood feeders) Microcerotermes parvus 12.750 (n = 2) Microtermes sp. nr. grassei 20.140 (n = 2) Nasutitermes latifrons 10.320 (n = 2) Protermes prorepens 18.600 (n = 2) Schedorhinotermes putorius 20.520 (n = 2) (Soil/wood interface feeders) Amalotermes phaecocephalus 24.517 (n = 2) Cephalotermes rectangularis 14.770 (n = 2) Termes hospes (Soil feeders) Apilitermes longiceps Astalotermes quietus Coxotermes bukokensis Crenetermes albotarsalis Cubitermes fungifaber Cubitermes heghi Jugositermes tuberculatus Labidotermes sp. nov. 1 Labidotermes sp. nov. 2 Ophiotermes grandilabius Pericapritermes amplignathus Procubitermes arboricola Thoracotermes macrothorax

13.890 ± 3.030 5.470 (n = 2) 10.300 ± 2.480 6.150 (n = 2) 3.84 ± 0.56 6.020 ± 2.53 3.810 ± 0.71 4.320 (n = 2) 6.040 ± 0.31 4.220 (n = 2) 8.420 ± 2.67 10.990 ± 0.67 5.090 ± 0.790 5.650 ± 0.960

0.079 0.141 0.212 0.334

± ± ± ±

0.021 0.037 0.036 0.115

0.125 0.081 0.533 0.746

0.025 0.029 0.193 0.558

0.069 (n = 2) 0.191 ± 0.027 0.253 (n = 2)

— — —

1.10 ± 0.01 (n = 4) 1.10 ± 0.01 (n = 4) 1.13 ± 0.15 (n = 4)

0.040 (n = 2) 0.061 (n = 2) 0.163 (n = 2) 0.030 ± 0.011 0.094 (n = 2)

— — — — —

1.10 ± 0.01 (n = 1.07 ± 0.09 (n = 1.11 ± 0.03 (n = 1.07 (n = 2) 1.12 ± 0.11 (n =

0.052 (n = 1) 0.252 (n = 2) 0.798 ± 0.172 (n = 13)

— —

1.10 (n = 1) 1.08 ± 0.01

—

1.33 ± 0.15

0.167 (n = 2) 0.205 ± 0.071 0.274 (n = 2) 0.171 (n = 2) 0.263 ± 0.050 0.302 ± 0.047 0.229 (n = 2) 0.265 (n = 1) 0.189 (n = 2) 0.147 ± 0.092 0.319 ± 0.079 0.325 ± 0.022 0.242 ± 0.057

— — — — — — — — — — — — —

1.31 1.24 1.32 1.40 0.94 1.29 1.31 1.13 1.21 1.65 1.04 1.40 1.09

± ± ± ± ± ± ± ± ± ± ± ± ±

4) 4) 4) 8)

0.16 0.68 0.07 0.13 0.19 0.15 0.08 0.03 0.04 0.18 0.14 0.18 0.15

n.d., not determined. 1 n = 3–10, except where stated. 2 Corrections made to Warburg manometer readings for CH4 and H2 where fluxes are known (data presented are RQs); otherwise corrected for CH4 only (data presented are for RQapp). 3 Compiled from measurements of acetylene reduction, assuming C2H2 has three times the affinity for nitrogenase than N2. Rates are (␮mol g−1 h−1): Coptotermes acinaciformis, 0.232; Nasutitermes walkeri, 0.051; Reticulitermes flavipes, 0.068. 4 Data from Williams et al. (1994); all gas fluxes by chromatography. 5 Data from Odelson and Breznak (1983); O2 and CO2 by manometry. 6 Effluxes determined by Anklin-Mu¨hlemann et al. (1995); RQ of M. subhyalinus determined by Veivers et al. (1991). For an explanation of groupings, see text. MAJ = major worker; MIN = minor work.

larger termites) in flasks often showed exceptional RQapp values (⬍ 0.70, ⬎1.50), which are difficult to interpret physiologically or biochemically (but see below). Methane production, on a weight-specific basis, was not affected by crowding in R. lucifugus (P⬎0.10, data not shown), but in T. hospes, a larger methane efflux occurred when large numbers of termites (⬎100 per flask) were used (Fig. 3, r2 = 0.684, P ⬍ 0.05). T hospes

is a strongly methanogenic species (Bignell et al., 1997; see also Table 1). The increased production in crowded flasks could reflect the effects of enhanced CO2 concentration in the flask atmosphere, acting through a methanegenerating mechanism with first-order kinetics, but other explanations are possible, for example that decreased O2 concentrations may influence the electron flow in termite hindguts.

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L. NUNES et al. 1.5

Methane efflux (µmol g -1 h -1)

1.2

1.4

RQ

1.3 1.2

1.1

1.1 1.0 0.9 0.8 0.7 0.6

1.0

0.5 0 0.9

60

90

120

150

180

210

240

Time (min)

FIGURE 1. The relationship between time of incubation and RQ (monometry corrected for CH4 and H2) in Reticulitermes lucifugus, determined under laboratory conditions at 27°C. The RQs shown are determined at 30-min intervals, but excluding the first 30 min of incubation. The first data shown are therefore for the 30–60-min period of incubation. n = 13 (determinations). Mean ± 1 SD is shown. 1.4 B B

1.3 B

B

BB

B

1.2

B

RQ

B B

B B

B B

1.1

B B B

1.0 B

0.9 0

100

200

300

Weight of termites (mg)

FIGURE 2. The effect of crowding (as live weight of termites) on the RQ of Reticulitermes lucifugus, under laboratory conditions at 27°C. Termites were incubated for 4h in identical Warburg flasks of mean volume 14.70 ± 0.65 ml (23 flasks). n = 18 (determinations).

Accounting for unknown H sinks (H2 efflux and N2 fixation) The oxygen required to oxidize carbohydrate to CO2 and H2O with allowances, in a balanced equation, for the observed loss of some C as CH4 and some H as H2 gas, as CH4 gas or as the initial product of N2 fixation (NH3) can be calculated (theoretical O2 consumption). The calculated and observed consumptions of O2 then can be compared. Any discrepancy (i.e. observed xO2 ⬍ theoretical xO2) indicates the possible presence of a hydrogen sink (or sinks) not requiring the consumption of O2, in addition to the observed formation and evolution of CH4 and H2 (or CH4 only, where H2 is unknown). This is useful in that it permits, in species where only data for exchanges of O2, CO2 and CH4 are available, the interpretation of RQapp values that are in excess of 1.00 by providing an estimate of the size of the sink (as reduc-

100

200

300

Weight of termites in flask (mg)

FIGURE 3. The effect of crowding (as live weight of termites) on the emission of methane by field-collected Termes hospes at 26°C. Termites were incubated for 3h in sealed 100-ml gas-sampling vials. n = 9 (determinations).

ing equivalents, ␮mol H2) required to reconcile the oberved RQ (RQapp) with the use of carbohydrate as the substrate for respiration. For Group I species, the known effluxes of CO2, H2 and CH4, together with the known rates of N2 fixation, give values for theoretical O2 consumption that would produce the following RQs: Coptotermes acinaciformis, 1.02; Nasutitermes walkeri, 1.03; Reticulitermes flavipes, 1.05. For the last two species, these are sufficiently close to the observed values (1.05 and 1.03, respectively) to be consistent with carbohydrates being the main substrates of respiration, with CH4, H2, NH3 and H2O (from aerobic metabolism) as hydrogen sinks. Similarly, in Macrotermes subhyalinus (Group II), the RQ calculated on the same basis is 1.01, virtually identical to the observed value of 1.00. Although the rate of N2 fixation has not been measured in this species, its effect on the observed RQ is evidently small. In the remaining species in Group II (Reticulitermes lucifugus and Reticulitermes santonensis) and the majority of those in Group III (for which only CH4 efflux data are available), there is a greater difference between observed RQ (RQapp) and that expected from the known gas exchanges, ranging from 0.06 (Microtermes grassei, Cephalotermes rectangularis) to as much as 0.62 (Ophiotermes grandilabius). However, in Cubitermes fungifaber, which, as noted above, has a low observed RQ of 0.94, the expected value is 1.05. In Pericapritermes amplignathus, the observed and expected RQ are essentially the same. For Group III species, the question arises as to whether the production of H2 gas or the fixation of N2 could account for the (generally large) difference between RQapp and that expected from the calculation of expected O2 consumption. The issue can be partly addressed by the simple computation set out in Table 2 for a selection of species. Here, the actual O2 consumption (from Warburg manometry) is subtracted from the calculated consumption (Step 3 above). In all cases, except C. fungi-

RESPIRATORY QUOTIENT OF TERMITES

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TABLE 2. Calculation of the size of the hydrogen sink, as reducing equivalents (␮mol H2) required to account for the difference between observed and theoretical O2 consumption (terms defined in the text) in 15 species for which only O2, CO2 and CH4 flux data are available Observed O2 consumption, ␮mol g−1 h−1

Species Macrotermes mulleri minors Microcerotermes parvus Nasutitermes latifrons Schedorhinotermes putorius Cephalotermes rectangularis Termes hospes Apilitermes longiceps Astalotermes quietus Coxotermes bukokensis Cubitermes fungifaber Cubitermes heghi Ophiotermes grandilabius Pericapritermes amplignathus Procubitermes arboricola Thoracotermes macrothorax

10.090 12.750 10.320 20.520 14.770 13.890 5.470 10.300 6.150 6.020 3.810 8.420 10.990 5.090 5.650

Theoretical O2 consumption, ␮mol g−1 h−1

Deficit of reducing equivalents (as ␮mol H2)

10.802 13.930 11.307 23.046 15.718 17.259 6.963 11.125 7.856 5.727 4.758 14.389 10.961 6.595 5.980

1.424 2.360 1.974 5.052 1.896 6.738 2.986 1.650 3.412 − 0.586 1.896 11.938 − 0.058 3.010 0.660

Theoretical O2 consumptions are derived as shown in the text on the assumption that carbohydrate is the substrate supporting respiration.

faber, this product has a positive value that, when doubled, gives the number of moles of reducing equivalents as ␮mol H2 (1 mol of O2 requiring 2 mol of H2 to form 2 mol of H2O) required to give the observed and expected RQ the same value, on the assumption that carbohydrate is the respiratory substrate. Thus, to explain the observed RQ on the basis of H2 gas production, most species would need to produce more than 1 ␮mol g−1 h−1, and some up to 10 × or more that amount. Actual measurements of H2 effluxes from termites are few in the contemporary literature, but our highest value of 0.746 ␮mol g−1 h−1 (for R. santonensis) is at the upper end of the range reported from analyses with modern equipment (e.g. Odelson and Breznak, 1983; Williams et al., 1994; Anklin-Mu¨hlemann et al., 1995). Similarly, to explain the observed RQ on the basis of N2 fixation would require most species to be consuming at least 0.333 ␮mol N2 g−1 h−1 and, in some cases, much more. Reported rates of N2 fixation in termites vary widely, sometimes even within the same species, but the highest of which we are aware is about 0.5 ␮mol g−1 h−1 (e.g. Breznak et al., 1973). Most other reports give much

lower rates (e.g. French et al., 1976; Prestwich et al., 1980; Bentley, 1984; Lovelock et al., 1985; Waller et al., 1989). DISCUSSION

Whereas RQ is not a proof of the identity of particular substrates used in respiration, it is, nevertheless, an index of the processes occurring and, if determined with sufficient accuracy in conjunction with determinations of CH4 and H2 fluxes, should illuminate the nature of basic respiratory metabolism. The data presented provide evidence that, with few exceptions, termite RQs are at or above 1.00, and therefore consistent with the conclusion that carbohydrate is the substrate utilized in respiration. Soil-feeding species are notable in showing measured values in the range 1.09–1.65 (the exceptions, Cubitermes fungifaber and Pericapritermes amplignathus, are considered above). Such results are difficult to reconcile with previous reports that give RQ values for termites below 1.00, unless the earlier work, largely based on manometry but

TABLE 3. Illustration of the effects of H2 efflux on apparent RQ, with and without correction of Warburg manometric data for CH4 production RQ

RQapp (Warburg) Not corrected

C6H12O6 + 5H2O C6H12O6 0.25CH4 C6H12O6 4H2O

+ 5O2→5.5O2 + 0.5CH4 + 5O2→5.75CO2 + + H2 + 4.5H2O + 5O2→6CO2 + 2H2 +

Corrected for CH4

1.10

1.22

1.10

1.15

1.53

1.44

1.20

2.00

2.00

The scenarios assume that carbohydrate is the substrate for respiration and that one-sixth of the electrons are routed either towards CH4, H2, or equally divided among the products.

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not employing the Warburg system, has been prone to large errors. Measurements made using gas chromatography generally give RQs at or slightly above 1.05 (e.g. Odelson and Breznak, 1983; Williams et al., 1994), but this method may not be practical under field conditions. As the Warburg system is no longer manufactured commercially, it is difficult to obtain the range of flask sizes necessary to determine the optimum conditions for termite incubations. Hence, the size of the flask (13–16 ml) and the biomass of termites (ca. 150 mg) employed in the present study were somewhat arbitrary and we do not know whether incubations on a larger scale would produce significantly different results. The only reported attempt to determine the RQ of termites within their mounds is that of He´brant (1970), but the disadvantage of this approach (other than practical difficulties) is that no distinction can be made between the metabolism of termites and that of microorganisms present in the mound materials. A recent study of 13 nearctic species by Wheeler et al. (1996) has shown that respiratory gas fluxes of termites can be affected by the ambient concentrations of O2 and CO2. After 48h of incubation in a closed vessel also containing food materials (in which CO2 accumulated to 4–8% and O2 declined to 16–18%, conditions arguably typical of the interiors of nests and galleries), some species reduced their weight-specific rates of O2 consumption and CO2 production by as much as twothirds. However, the effect was not universal, nor did O2 and CO2 fluxes necessarily respond in the same way within each species. Clearly, a tendency for CO2 output to decline as ambient concentrations increased (or O2 decreased) without a corresponding fall in O2 consumption could account for the fall of RQ with time of incubation, but this does not invalidate the inferences that can be drawn, concerning the substrates utilized for respiration. A conclusion that carbohydrates are the substrates utilized in respiration in almost all species examined calls into question how the various evidences that lignin can be degraded in the termite intestine [reviewed by Breznak and Brune (1994)] should be viewed. The longstanding objection that no enzymatic mechanism was known that could break down lignin under anaerobic conditions (see Zeitkus, 1983; Reddy, 1984) is no longer relevant, since it is now established that O2 is readily available in a substantial part of the hindgut (Brune et al., 1995a). The following equation shows that the complete oxidation of lignin, calculated as coniferyl units, would give an expected RQ of 0.87: C10H12O3 + 11.5O2 = 10CO2 + 6H2O. Calculated as vanillin (C8H8O3), the expected RQ is 0.94, as coumaric acid (C9H8O3), it is 0.95, and as ferulic acid (C10H10O4), 0.95 (equations not shown). Of the aromatic acids normally considered to be included in the polymerization process producing lignin (Pearl, 1967),

only syringic acid (C9H10O4) would give an expected RQ of 1.00 upon complete oxidation. The ‘idealized lignin molecule’ depicted by Breznak and Brune (1994) would be oxidized according to the following scheme: C14H154O44 + 158.5O2 = 142CO2 + 77H2O, for which the expected RQ is 0.90. If the degradation of either lignin or lignin-derived polyaromatic materials in soil organic matter occurs extensively, this would be expected, therefore, to reduce RQ below 1.00. Based on the quantitative study of 14C-lignin degradation in woodfeeding nasute species Nasutitermes exitiosus carried out by Cookson (1988), Slaytor et al. (1997) have estimated that if lignin were oxidized in Nasutitermes walkeri at the same rate, then 0.51 nmol termite−1 h−1 of lignin (calulated as coniferyl units) would be oxidized. The effect of this would be to reduce the observed RQ by about 0.02 to 1.01, assuming that polysaccharide is used to meet the remaining respiratory needs and that other gas exchanges are as observed. By contrast, there is sufficient cellulase activity in the gut to produce 21 nmol of glucose termite−1 h−1 (Schulz et al., 1986) and sufficient xylanolytic activity to generate xylose at the rate of 6 nmol termite−1 h−1 (Hogan et al., 1988), more than enough carbohydrate substrate to support the measured rate of respiration. The position of Coptotermes acinaciformis (mean RQ = 0.94 ± 0.14) is anomalous, and on the basis of RQ data alone (at 25°C), the degradation of lignin in significant quantities cannot be excluded. However, at 30°C, the mean RQ rises to 1.07 ± 0.15 (Lo, unpublished). Whereas there is evidence that C6–C1 and C6–C3 aromatic compounds can be oxidized in termites, including both wood- and soil-feeding forms (Brune et al., 1995b), the manometric evidence clearly points to carbohydrate as the principal respiratory substrate, and the issue of lignin/polyaromatic compound degradation in termites remains unresolved. Close inspection of the gut contents of soil-feeding termites shows that fragmented plant tissues and macerated wood fibres are present, in addition to soil organic matter and other materials (Sleaford et al., 1996). Hence, polysaccharides are almost certainly available as substrates in soil-feeders, as in all other termites. Evidence that carbohydrate is the principal substrate for respiration in all species eliminates the need to propose a novel mechanism of lignin/polyaromatic compound cleavage unique to termites. Further, it assists the understanding of higher termite phylogeny, as it more easily explains the independent acquisition of the soil-feeding habit in at least four lines of evolution (Noirot, 1992; Bignell, 1994), there being no need to postulate a radical change in basic digestive organization on each occasion. The same argument suggests that the low RQ in Coptotermes acinaciformis and low RQapp in Cubitermes fungifaber should be regarded as aberrant, possibly the consequence of stress under the conditions of incubation, although a novel digestive process remains possible.

RESPIRATORY QUOTIENT OF TERMITES

In many cases, there was a discrepancy between the observed and the expected RQ (observed O2 consumption ⬍ calculated O2 consumption), suggesting the existence of a sink or sinks for hydrogen in addition to the production of water and methane. Such sinks might be the reduction of atmospheric N2 to NH3, a process known to occur in some termites (Breznak et al., 1973; Beneman, 1973) or the production and accumulation of organic acids (e.g. acetate, formate). Acetogenesis from CO2 is known to be an important process in termite intestines (Brauman et al., 1992; Breznak and Brune, 1994), but it is usually assumed that the acetate generated is utilized aerobically by the termite host, in which case, at steady state, the molar ratio of CO2 produced to O2 utilized in acetate metabolism would be 1.0, and no effect on RQ would be expected. However, if short chain fatty acids were formed in the hindgut as part of a sink mechanism and subsequently voided with faeces (without being oxidized by microorganisms), a substantial elevation of observed RQ should be expected. Kuhnigk et al. (1996) have recently investigated the metabolism of sulphatereducing bacteria (genus Desulfovibrio) isolated from termite guts. Amongst other reactions, the organisms were able to utilize H2, producing H2S. Although the model presented by Kuhnigk et al. assumes that this product is subsequently oxidized back to sulphate (by O2) to alleviate any toxic effects, in effect creating a sulphur cycle within the gut, the possibility that H can be removed by these reactions without the requirement for O2 consumption seems worth investigating. The question arises as to whether any known biochemical mechanism in termites can explain values of RQapp in excess of about 1.20. There is a possibility that the high values of RQapp in most soil feeders can be explained by massive N2 fixation. However, the recent study by Tayasu et al. (1997) of nitrogen stable isotope ratios in the food materials and body tissues of five species from the Cameroon forest assemblage (Cubitermes heghi, C. fungifaber, Thoracotermes macrothorax, Astalotermes quietus and Jugositermes tuberculatus) suggests that fixation of atmospheric N2 is very low or absent. Hydrogen efflux, therefore, remains the most likely explanation of the high values of RQapp observed. This can be demonstrated in a comparison of real and apparent RQs in three scenarios where one-sixth of the electrons from carbohydrate oxidation are routed either towards CH4, H2, or equally divided between these products (Table 3). Where both CH4 and H2 are produced as H sinks, arguably likely in soil-feeding species, correction of manometric data for CH4 (but not for H2) still produces an RQapp well in excess of 1.00. From these arguments, the production of quite large amounts of H2 is predicted for termite species with a high RQapp. REFERENCES Anklin-Mu¨hlemann R., Bignell D. E., Veivers P. C., Leuthold R. H. and Slaytor M. (1995) Morphological, microbiological and biochemical studies of the gut flora in the fungus-growing termite

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Acknowledgements—Most of the work described was carried out at the Humid Forest Station of International Institute of Tropical Agriculture, Mbalmayo, Cameroon and in the Unit of Timber Technology, Imperial College, London. We acknowledge the financial support provided by the Natural Environment Research Council (UK) through its Terrestrial Initiative in Global Environmental Research Programme, award no. GST/02/625. We thank Michael Slaytor and Andreas Brune for discussions on the interpretation of manometric data and Prof. Corinne Rouland for methodological advice.