IRG/WP 95-10136 Document No. IRG/WP 95 - THE INTERNATIONAL RESEARCH GROUP ON WOOD PRESERVATION SECTION 1 Biology The influence of boric acid on respiratory quotients and methane production of subterranean termites L.Nunest.2 &D. J. Dickinson 1 1 Department of Biology, Imperial College, London SW7 2BB, UK 2 Timber Division, Lab. Nacional de Engenharia Civil, 1799 Lisboa Codex, Portugal Paper prepared for the 26th Annual Meeting Helsingér, Denmark June 1995 IRG Secretariat Box 5607 ‘$-114 86 Stockholm ‘SwedenThe influence of boric acid on respiratory quotients and methane production of subterranean termites L. Nunes!.2 & D. J, Dickinson’ 1 Department of Biology, Imperial College, London SW7 288, UK 2 Timber Division, Laborat6rio Nacional de Engenharia Civil, 1799 Lisboa Codex, Portugal ABSTRACT The toxicity of boron compounds to subterranean termites (Reticulitermes lucifugus) was studied by analysing changes in the levels of oxygen uptake, fespiratory quotients and methane production. Termites were fed with filter paper treated at different levels of boric acid, the RQ was evaluated by constant volume manometry and the methane production by gas chromatography. Results are discussed in relation to termite gut symbiontes. KEYWORDS Subterranean termites, Boron, Respiration, Methane INTRODUCTION Borates have been used for a long time as wood preservatives for timber, either protected or semi-protected, and in combination with other active ingredients, for timbers in more severe situations. Although this type of products were often viewed as ‘ineffective’ because boron could not be fixed and was not able to protect timber in ground contact (Williams, 1990), there is a renewed interest in boron as a wood preservative as a result of an increased need to find more ‘environmentally friendly’ preservative systems (Reiner, 1993). There is a general acceptance of the efficacy of boron towards fungi and insects (Dickinson & Murphy, 1989), in particular, boric acid and boron salts are considered toxic to termites (Williams & Amburgey, 1987, Drysdale, 1994) Nevertheless, the efficacy against all species of termites is still not well established, for instance, Gay and co-workers (1958) suggested that boron compounds have amuch greater effect on the group of termites with intemal protozoa, mostly the so called lower termites (O'Brien & Slaytor, 1982), than those without. The exact mechanism of their toxicity is still not well understood, though some data is starting to be available. Williams and co-workers (1990) showed that feeding termites with low levels of boron affects their digestive processes and the vitamin 812 content, but the reason for this remained undifined. Grace and co-workers (1992) verified the susceptibility of three species of protozoa present in the termite gut, after forced feeding of the subterranean termite Coptotermes formosanus with increasing concentrations of disodium octaborate tetrahydrate, and found that the susceptibility of the protozoa appeared to be directly proportional to their size and location in the hindgut. The smallest and least susceptible species is usually found in the posterior part of the hindgut. Taking also into account that, termite mortality is not the direct result of starvation due to the reduction in protozoa numbers, for at least 30 days (Khoo & Sherman, 1979) and sometimes for longer periods (personal observation), it can not be determined whether the boron compound was directly toxic to the protozoa or whether they were affected secondarily as a result of the boron toxicity to the termite. In order to further study the interaction between boron compounds and termites, an attempt was made to evaluate the effect of boric acid on the respiration (0, uptake, CO, production and respiratory quotients) and on the methane production of a subterranean termite, Reticulitermes lucifugus. MATERIALS AND METHODS Termites were fed with filter paper treated at different levels of boric acid and the RQs were evaluated by constant volume manometry using a Warburg apparatus. ‘A number of termites was placed in a convenient flask and a 20% solution of KOH was added to the central well whenever necessary (measurement of the Op uptake). The flasks were attached to the manometer and placed on a water bath fixed at 27.0 °C + 0.5°C. After 60 minutes of equilibrium time the readings were started. The manometer fluid was defined based on the Krebs manometer fluid (Umbreit, 1972) which was in itself a variation of Brodie's manometer fluid (Dixon, 1952). The results were obtained at 30 minutes intervals for 240 minutes and expressed in nl per mg of termite wet weight per hour. In what concems the methane production, 30 termites were fed as described and then sealed in glass vials (25 mi) containing a small piece of filter paper that provided a firm substrate to the insects.After different periods of incubation, a 0.5 mi sample of the 25 mi headspace was taken with a precision syringe. All measurements were made at least in triplicate. Preliminary experiments showed that methane production was linear for at least 4 hours in the sealed tubes. These periods of incubation did not seem to effect the termites in any visible manner. Methane in headspace samples was measured using a Pye Unicam gas ‘chromatograph with a Porapack R column, operated at room temperature and equipped with a flame ionization detector and a Hewlett Packard 3390 A Integrator. An external methane standard (100 ppm) was used and the results were transformed to a rate of methane emission in ppm per mg of termite wet weight per hour. RESULTS RESPIRATION Figure 1 shows the relation between the number of termites on the flasks (without treatment), expressed as wet weight, and the respiratory quotients obtained, wet weight (mg) Figure 1: A. lucifugus. RQ versus wet weight of termites in the flask In the following tables | to III the results obtained (n=number of replicates) for the respiration parameters are presentedTable |: R. lucifugus. Untreated termites. Results of constant volume manometry wet weight of O,uptake | CO, production termites (mg) (uimgih) (ulmgmn) RQ 40 - 100, 0.38 (0.01) 0.47 (0.02) 1.24 (0.07) (n=10) 101 - 240 0.39 (0.01) 0.43 (0.03) 1.12 (0.06) (n=10) total (n=20) 0.38 (0.01) 0.45 (0.02) 1.18 (0.06) ‘andard Gaviaion brackets Table II: F. lucifugus. Starved termites. Results of constant volume manometry number of, Q, uptake | CO, production RQ starving days (ulimgih) (ulimg/h) 7 0.45 (0.07) 0.45 (0.08) 1.02 (0.10) = @ BO erie por Wak Standard devon wh Brackets Table Ill R. lucifugus. Boric acid treated termites, Results of constant volume manometry time of boric acid | exposure | O,uptake | CO, production RQ (%BAE in paper) | _ (days) (uVingih) (ulimgih) 0.04 (n=10) 3 0.51 (0.01) 0.52 (0.03) 1.05 (0.06) 0.08 (n=10) 3 0.46 (0.04) 0.52 (0.08) 1.20 (0.20) 0.16 (n=5) 3 0.37 (0.01) 0.58 (0.04) 1.61 (0.23) 0.32 (n=5) 3 0.44 (0.02) 0.56 (0.04) 1.31 (0.16) 0.64 (n=5) 3 0.52 (0.04) 0.56 (0.02) 1.08 (0.06) ‘D tertes per Wask,slandard Geviaon bracketsMETHANE PRODUCTION Figure 2 shows the relation between the number of termites on the flasks (without treatment), expressed as wet weight, and the methane production obtained. 03 5 | 3? 02 fe. | ge . | FBa ae | ol — 0 ee wet weight (mg) Figure 2: R. lucifugus, Methane emission versus wet weight of termites in the flask In the following tables IV to VI the results obtained for the methane emission are presented (n = number of replicates). Table IV: R. lucifugus. Untreated termites. Results of methane emission Wet weight of termites methane emission (mg) (ppm /mgmn) 21 - 60 (n=10) 0.20 (0.03) 61 - 100 (n=10) 0.17 (0.04) 101 - 240 (n=10) 0.19 (0.03) total (n=30) 0.19 (0.04) Tandard deviation brackets Table V: R. lucifugus. Starved termites. Results of methane emission number of starving methane emission days (ppmimg/h) 5 0.06 (0.01) 1726) standard deviaionw breckalaTable VI: A. lucifugus. Boric acid treated termites. Results of methane emission Boric acid methane emission (% BAE in paper) (ppm/mg/n) 0.02 0.12 (0.03) 0.04 0.13 (0.03) 0.06 0.06 (0.03) 0.08 0.09 (0.03) o4 0.04 ( 0.03) =o Standard deviaion h brackets DISCUSSION As initially expected and with results comparable to data already existent on other wood feeding termite species (Brauman, et al, 1992), there was a considerable amount of methane being produce by the termites, A decrease in this production of methane, was verified for the termites fed with boric acid treated paper. The same pattern had already been registered for A. flavipes when fed with disodium octaborate tetrahydrate (Williams et al, 1990). It is, therefore, assumed that the boron is killing the methanogenic bacteria although the actual mechanism for this to happen was not assessed. In what concems the respiration data, an average RQ of 1.18 was obtained for untreated termites and this was close to what had been anticipated, taking into account that some level of fermentation was taking place in the hindguts (Bignell & Anderson, 1980). After a starving period of seven days the symbiont content of the gut is severely reduced (Yoshimura, et al,1994), hence the average RQ registered that was close to 1.00. There was a surprising increase on the RQs when certain levels of boric acid were added to the termites food source. The explanation for this effect is still not found,Nevertheless, in what concerns all the respiration data, the results should be considered with some care, taking into account the possible influence of the ‘substrate used to maintain the termites and also the volume of the recipient used to make the measurements. Hébrent (1970), also showed that the oxygen uptake of the termites was influenced by the substrate they were maintained on, namely filter paper as in the present study. Yet, a RQ of 1.49 was reported by Benedict & Lee (1937) for over-fed geese and this was explained by a possible change on the normal metabolism of the animals with an increase on the production of lipids. ACKNOWLEDGEMENTS The authors wish to acknowledge The British Council for financial support and Laboratério Nacional de Engenharia Civil for the study leave given to Ms. Lina Nunes, to undertake the research project. We also wish to thank Dr. Roger Berry of the Building Research Establishment, Watford, for the supply of the termites used in this work. REFERENCES Benedict, A. & Lee, T. 1997. Lipogenesis in the animal body with special reference to the physiology of the goose. Carnegie Inst. Wash. Public. n? 489: 1-232. Bignell, D. E. & Anderson, J. M. 1980, Determination of pH and oxygen status in the guts of lower and higher termites. J. Insect Physiol. 26: 183-188, Brauman, A. ef al. 1992. Genesis of acetate and methane by gut bacteria of nutritionally diverse termites. Science 257: 1384-1387. Dickinson, D. J. & Murphy, R. J. 1989. Development of boron based wood preservatives. in Record of the 1989 Annual Convention of the Wood Preservation Association. British Wood Preserving Association, pp 35-44. Dixon, M. 1952. Manometric methods as applied to the measurement of cell respiration and other processes. Cambridge Univ. Press, Cambridge. 161 pp. Drysdale, J. A. 1994. Boron treatments for the preservation of wood - A review of efficacy data for fungi and termites. The Int. Res. Group on Wood Preservation Doc. n® IRG/WP 94-30037. IRG Secretariat, Stockolm. 21 pp. Gay, F. J. ef al, 1958. Laboratory studies of termite resistence. III. A comparative study of the anti-termite value of boric acid, zinc chloride and "Tanalith U". CSIRO Div. Ent. Tech, Paper n®4,Grace, J. K. et al. 1992. Resistance of borate-treated Douglas-fir to the Formosan subterranean termite. Forest Prod. J. 42 n®2: 61 - 65. Hébrant, F. 1970, Circadian rhythm of respiratory matabolism in whole colonies of the termite, Cubitermes exiguus. J. of Insect Physiology, 16: 1229-1235. Khoo, B. K. & Sherman. 1979. Toxicity of chloropyrifos to normal and defaunated Formosan subterranean termites. J. Econ. Entomol. 72: 298 - 304. Obrien, R. W. & Slaytor, M. 1982. Role of microorganisms in the metabolism of termites. Aust. J. Biol. Sci. 35: 239 - 262. Reiner, J. B. 1993. Borates as wood preservatives - an environmental , helth and safety perspective. 2 Intemational Symposium on Wood Preservation. The Int. Res. Group on Wood Preservation Doc. n® IRG/WP 93-5001, IRG Secretariat, Stockolm. pp 59-76 Umbreit, W. W. ef al. 1972. Manometric & Biochemical Techniques. Burgess Publishing Company, Minneapolis, Minnesota. 315 pp. Williams, L. H. 1990, Potential benefits of diffusible preservatives for wood protection: an analysis with emphasis on building protection. Proc. First International Conference on Wood Protection with Diffusible Preservatives. Forest Products Research Society. pp 29-34. Williams, L. H. & Amburgey, T. L. 1987. Integrated protection against Lyctid beetle infestations. IV. Resistance of boron-treated wood (Virola spp.) to insect and fungal attack. Forest Prod. J. 37, n®2: 10-17. Williams, L. H. et al. 1990, Borate-treated food affects survival, vitamin B12 content, and digestive processes of subterranean termites. The int. Res. Group on Wood Preservation Doc. n® IRG/WP/1448. IRG Secretariat, Stockolm. 16 pp. Yoshimura, T, et al. 1994. Effects of starvation on the protozoan fauna in the hindgut of Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Jpn. J. Environ. Entomol. Zool. 6 (1): 31-36.