The role of termite CH4 emissions on ecosystem scale: a case study in the Amazon rain forest

The magnitude of termite methane (CH4) emissions is still an uncertain part of the global CH4 budget and current emission estimates are based on limited field studies. We present in-situ CH4 emission measurements of termite mounds and termite mound sub samples, performed in the Amazon rain forest. Emissions of five termite mounds of the species Neocapritermes brasiliensis were measured by use of a large flux chamber connected to a portable gas analyser, measuring CH4 and CO2. In addition, the emission of mound sub samples was measured, after which termites were counted, so that a termite CH4 and 5 CO2 emission factor could be determined. Mound emissions were found to range between 17.0 -34.8 ✿✿✿ and ✿✿✿✿ 34.8 nmol mound s for CH4 and between 1.6-13.5 ✿✿ 1.1 ✿✿✿✿ and ✿✿✿✿ 13.0 μmol mound s for CO2. A termite emission factor of 0.32 ✿✿✿✿ 0.35 μmol CH4 g −1 termite h −1 was found, which is ✿✿✿✿✿✿ almost twice as high as the only other reported average value for the Amazon. By combining mound emission measurements with the termite emission factor, colony sizes could be estimated, which were found to range between 55-125 thousand individuals. 10 Estimates were similar to literature values, and we therefore propose that this method can be used as a quick non-intrusive method to estimate termite colony size in the field. The role of termites in the ecosystems CH4 budget was evaluated by use of two approaches. Termite mound emission values were combined with local termite mound density numbers, leading to an estimate of 0.15-0.71 nmol CH4 m s on average emitted by termite mounds. In addition, the termite CH4 emission factor from this study was combined with termite density 15 ✿✿✿✿✿✿✿ biomass numbers, resulting in an estimate of termite emitted ✿✿✿✿✿✿✿✿✿✿✿✿✿ termite-emitted CH4 of ∼1.0 nmol m s. Considering the relatively low net CH4 emissions previously measured at this ecosystem, we expect that termites play an important role in the CH4 budget of this Terra Firme ecosystem.

gases, but its natural sources are still not well understood. Anaerobic decomposition processes in wetlands are expected to represent the largest natural CH 4 source, but estimates remain a large source of uncertainty (Kirschke et al., 2013;Saunois et al., 2020). Recently, alternative CH 4 production mechanisms and their possible important role on ecosystem scale have been proposed, such as the CH 4 production by living vegetation (Bruhn et al., 2012;Wang et al., 2014), the CH 4 emission due 25 to photo and thermal degradation (Lee et al., 2012), or the transport of anaerobic soil-produced CH 4 through wetland trees (Pangala et al., 2015;Rice et al., 2010). An additional known CH 4 source in tropical ecosystems is the emission by termites.
Termites (isoptera) can mostly be found between 45 • N and 45 • S, and are especially abundant in warm ecosystems (Bignell, 2006;Brian and Brian, 1978;Gomati et al., 2011;Wood, 1988). They are highly socialised insects, living in large communities of up to several million individuals (Wood, 1988). Termites are considered 'ecosystem engineers': they are known for 30 decomposing organic substances, and moving and mixing organic and mineral materials, thereby enhancing humus formation, modifying soil structure, and improving soil fertility (Bignell, 2006;Brian and Brian, 1978;Bignell and Eggleton, 2000;Mishra et al., 1980;De Bruyn and Conacher, 1990;Wood, 1988). In addition, they are able to modify their environment to their needs: most termite species live in complex above or (partly) below-ground nests where temperature and moisture remain stable (Bignell, 2019;Noirot and Darlington, 2000;Wood, 1988 (Ashton et al., 2019).
Three main groups of termites can be distinguished, based on their main feeding habits: soil-feeding (humiverous) termites, who can mainly be found in and on the soil, decomposing decayed organic soil material, xylophagous termites, feeding on (decomposed) wood, which can also be found in living trees, and fungus-eating ✿✿✿✿✿✿✿✿✿✿✿✿ fungus-feeding termites, which live in a symbiotic 40 relationship with fungus (Eggleton, 2000;Sanderson, 1996).
CH 4 production by termites was first described and measured by Cook (1932). Follow up studies found that methane is produced by almost all termite species, and that its production takes place in the termite gut: in higher termites (dominant in tropical forests, more evolved species with respect to diet and community complexity) CH 4 production is caused by symbiotic 45 bacteria, and in lower termites the production is caused by flagellate protozoa (Bignell et al., 1997;Brune, 2018;Lee et al., 1971). In a laboratory experiment Zimmerman et al. (1982) measured the emission strength of individual termites and, by use of termite biomass estimates ✿✿✿✿✿✿✿ numbers, presented a global termite emission estimate of 150 Tg CH 4 yr −1 , which was estimated to be 40% of the global natural CH 4 emissions. Different estimates followed, resulting in lower estimates ✿✿✿✿✿ values, such as by Seiler et al. (1984) of 2-5 Tg yr −1 , by Fraser et al. (1986) of < 15 ✿✿ 14 ✿ Tg yr −1 , by Khalil et al. (1990)  were often performed alongside CH 4 emission measurements, and often ✿✿✿✿✿✿✿✿ generally a clear relationship between CH 4 and CO 2 emissions was found, of which the ratio is expected to be species dependent Jamali et al., 2013). For termite emitted  , and 3.5 Gt ✿✿ Pg yr −1 (Sanderson, 1996) (1 Gt ✿✿ Pg= 1000 Tg). In addition, Khalil et al. (1990) observed mound CO uptake and emissions, but reported them to be irregular and small. Strong termite mound N 2 O 70 emissions have also been detected (Brümmer et al., 2009b;Brauman et al., 2015), although they were also found to be very irregular or undetectable Zimmerman et al., 1982). Brauman et al. (2015) suggested that termite mound N 2 O emissions occur if N-rich organic matter is available.
In this paper, we are presenting a case study performed in a tropical rain forest in the Amazon, where we measured the emission of CH 4 and other gases of epigeal (above-ground) termite nests of the species Neocapritermes brasiliensis, a soil-feeding species 1 abundant in the Amazon (Constantino, 1992;Pequeno et al., 2013) (Dambros et al., 2016). In addition we measured the CH 4 emission of countable groups of termites. The goal of our research was twofold. Firstly, we are providing the first CH 4 and other gas emission measurements of the species N. brasiliensis, thereby expanding the limited literature on CH 4 emissions from Amazonian termites. Secondly, we are aiming to quantify the role of termite emissions in the CH 4 budget of this specific ecosystem, as part of a larger ecosystem CH 4 budget study (van Asperen et al., in preparation). In addition, we are presenting a possible quick non-intrusive field method to estimate termite colony size 95 in-situ.

Study site
The study was conducted at the experimental field site Reserva Biológica do Cuieiras -ZF2 (2 • 36" 32.67 S, 60 • 12"33.48 100 W, 40-110 m above sea level (a.s.l.), managed by the Instituto Nacional de Pesquisas da Amazônia (INPA), located ∼50 km northwest of Manaus (Brazil). Field site ZF2 consists of plateaus and valleys with typical terra firme forest with tree heights of 35-40 m on the plateaus and 20-35 m in the valleys. Soils on the plateau are clayey and can be classified as Oxisols and Ultisols. Soils in the valleys contain more sand and can be classified as Spodosols (Luizão et al., 2004;Zanchi et al., 2014).
The field site has a strong seasonality, with a wet season from December to April, and a dry season from June to September.

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Annual average temperatures range between 26-28 • C, and annual average precipitation is around 2400 mm. More information about the field site can be found in Araújo et al. (2002); Chambers et al. (2004); Luizão et al. (2004); Quesada et al. (2010); Zanchi et al. (2014). Measurements took place at the end of the wet season (March 2020).

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In the study area, two main trails exist, following the topography from valley to plateau, and termite nests in vicinity of these trails were inventoried. For practical reasons, only free-standing epigeal (above-ground) nests were considered, from here on called mounds. Twenty termite mounds were selected for further research, and of each mound the termite species was determined. For flux chamber measurements, five mounds with the same termite species were selected . For ✿✿✿ (nr. ✿✿✿ 13, ✿✿✿ nr.

14,
1 The species Neocapritermes brasiliensis is a wood/soil interface feeding species. Species feeding on extremely decomposed wood are in the centre of the 'wood-soil decomposition gradient' termite classification (Bourguignon et al., 2011), but are classified as soil-feeders according to Eggleton and Tayasu (2001).

✿
Of each mound, height and perimeter were measured. Termite mound volumes were estimated by use of the following formula, as also used in Ribeiro (1997) and in Pequeno et al. (2013): wherein V is the mound volume (cm 3 ), H is the height (cm), W is the width (cm), and T is the thickness (cm) of the mound.

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Termite mound surface was estimated by mathematically considering the lower part of the mound as a column, and the upper part as half a sphere. Details of each mound (dimensions, species, location) are given in Table 1.

Mound flux chamber set up
Collars (stainless steel, 15 cm height, 56.5 cm diameter) were placed around the five selected termite mounds a week before 125 the start of the measurements. Collars were inserted for approximately 5 cm into the soil/litter layer. In addition, one collar was placed at some distance from mound 15, containing only soil and litter, representing a blank (non-termite) measurement.
From here on, this collar will be referred to as 'blank measurement'.A flux chamber was created by use of a 220 L slightly cone-shaped ✿✿✿✿✿✿✿✿✿✿ polyethylene ✿ bucket, with a diameter of 57.5 cm. A strip of closed-pore foam (1 cm x 1 cm x 57.5 cm) was attached over the whole inner perimeter, so that if the bucket was placed on the collar, the foam strip would seal the part between 130 the bucket and the collar. Two one-touch fittings (1/4 inch, SMC Pneumatics) were installed on each side of the bucket. For chamber volume (CV), the termite mound volume (Table 1) was deducted from the bucket volume (220 L).

On
Fluxes could be calculated as follows: Mound adjacent soil and emissionsMound adjacent and soil emissions were measured around each mound once. For mound 13 and 14, this was done on the 2 nd measurement day, for mound 15 and 16, this was done on the 3 rd measurement day. Due to some practical issues, the measurements performed around mound 19 could not be used. Figure   N. brasiliensis has a relatively low soldiers:workers ratio of 1:100 (Krishna and Araujo, 1968 CH 4 and CO 2 emissions of 13 mound sub samples were measured. For each sub sample, the measured gas production was 245 plotted over the counted termites (Fig. 4). The fitted line has a forced intercept at y=0. For CH 4 , an emission of 0.0002985 nmol termite −1 s −1 was found (se=1.77*10 −5 ), fitted with an R 2 of 0.95 (n=13).  (Table 3). Population ✿✿✿✿✿ Colony ✿ size can also be estimated by use of mound volume or mound external surface. Table 3 shows the population ✿✿✿✿✿✿ colony ✿✿✿✿ size estimates, based on values as given by Lepage and Darlington (2000) for   (Noirot and Darlington, 2000)), explaining the wide range of reported termite mound CH 4 emissions (  Ho et al., 2013), and recent studies have been focusing on whether methanotrophic bacteria are also present in the termite guts, a topic still under discussion (Ho et al., 2013;Pester et al., 2007;Reuß et al., 2015). Different estimates exist on the effect of these bacteria on the net mound 285 flux. Sugimoto et al. (1998a) compared the δ 13 C of emitted by mounds to the δ 13 C of emitted by termites, and found a fractionation of 0.987 (emitted by mound/produced by termites). Other estimates range widely between no observable uptake to very strong uptake rates (up to 80%) Macdonald et al., 1998;Nauer et al., 2018;Sugimoto et al., 1998a) . A more elaborate overview of recent findings on termite mound uptake processes can be found in Nauer et al. (2018) and  (Ho et al., 2013;Khalil et al., 1990;Macdonald et al., 1998;Nauer et al., 2018;Seiler et al., 1984;Sugimoto et al., 1998a;Pester et al., 20 ✿ . The role of possible mound CH 4 uptake should also be acknowledged for the measurement of individual termite emissions ( Mound emissions ranged between 6-49 mmol mound −1 h −1 , which fits in the wide range of reported values (Table ??).

The
The relation between the amount of termites and emitted was found to be 82.2 µmol g −1 termite h −1 , which is higher than most reported values before. Also here it should be considered that mound material and termites were measured together.
Considering the presumably ongoing soil and mound material decomposition processes, the termite-produced emission rates are likely lower.

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The measured and emissions of individual mounds showed small variation, such as an emission increase of 25.3 to 29.5 nmol mound −1 s −1 at mound 15. One explanation is  (Jamali et al., 2011a;Ohiagu and Wood, 1976;Sands, 1965;Seiler et al., 1984). However, as our measured termite mounds are on the forest floor of a tropical rain In addition, since each mound measurement was performed at the same time of the day (±1 hour), it is unlikely that this variation is caused by a diurnal cycle. Another possibility is that the variation can be explained by the degree of air flow concentration drops. It is likely that even with a collar not all below-collar air flow was prevented, especially considering the depth and the porosity of the valley litter layer. This theory is supported by the overall coherent and concentrations during chamber closure, which followed the same pattern at all times (R 2 > 0.99). , variations. In case our set up was subject to minor air transport around ✿✿✿✿✿ below ✿ the collar, the given mound estimates will be an underestimation of  Jamali et al., 2013). Since mound 19 was located in a different part of the valley, it is likely that the characteristics of the surrounding organic matter were slightly different, affecting the /ratio, as also suggested by Seiler et al. (1984).

Colony size estimate
To estimate colony sizes of (epigeal) nest building termites, different methods exists. Excavation of a termite nest causes a strong disturbance, initiating an evacuation of the nest . To prevent this, fumigation with methyl bromide is usually applied, ✿✿✿✿ exist. can be removed from the nest debris by flotation in water, and can be counted. This process is labour intensive, and can take 335 five persons up to three weeks to finish one nest (Darlington, 1984;Jones et al., 2005). A faster method is by sub sampling known volumes of the mound, counting the termites in the sub sample, and extrapolating this to the total mound volume. Termite mounds can have irregular shapes, wherefore volume estimates strongly depend on which volume estimation approach (hemisphere, cone, column) is used (Jones et al., 2005).So while this method is faster and less intrusive, it depends strongly on correct volume estimation and it still takes several hours per mound to estimate a colony size.

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The population estimation method we tested combined CH 4 mound emissions with an in-situ measured  Pequeno et al. (2013) was quite weak (R 2 =0.41), and our estimates would fit in the general spread they observed in their data (Pequeno et al., 2013). Interestingly, Pequeno et al. (2013) concluded that mound volume is a weak indicator for population size for nests of the species N. brasiliensis, as also indicated by the weak correlation we found between mound volume and mound CH 4 emissions (Fig.??)..

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The influence of mound CH 4 uptake on our population estimate method should be contemplated ✿✿✿✿✿✿✿✿✿ considered: mound methanotrophic CH 4 uptake likely ✿✿✿✿✿✿✿ probably ✿ decreases the net mound CH 4 emission, resulting in an underestimation of the colony size when linking it to termite emission factors, as also suggested by Nauer et al. (2018). However, our termite emission factor was determined inside small pieces of undisturbed mound material, wherefore ✿ so ✿✿✿✿ that the materials CH 4 uptake rate was likely ✿✿✿✿✿✿✿✿✿ presumably ✿ only little affected. We hypothesise therefore ✿ It ✿✿ is ✿✿✿✿✿✿✿ therefore ✿✿✿✿✿ likely ✿ that our termite emission factor is underestimated 355 to the same degree as our mound emissions, wherefore both values can still be combined.
Overall, our colony size estimation approach can be considered as a test case for a quick population estimation method. The combination of one mound flux measurement (15 minutes) in combination with 5 sub sample measurements (5x5 minutes) can be performed within 1 hour, including the counting of the termites, being thereby ✿✿✿✿✿✿ thereby ✿✿✿✿✿ being faster than the original meth-360 ods. Also, the method is applicable to epigeal mounds of all species, independent of internal mound structure (Josens and Soki, 2010) or species characteristics (Pequeno et al., 2013). In addition, the population estimation method we present ✿✿✿✿✿✿ method is not strongly dependent on a correct mound volume estimate, which remains a source of uncertainty (Jones et al., 2005), and which has been shown to be a weak indicator of population size for some species (Pequeno et al., 2013;Josens and Soki, 2010 To estimate the role of termites on ecosystem scale, one approach is to combine mound emission values with termite mound density numbers. A local study reported a density value of 21.6 mounds ha −1 for the species N. brasiliensis specifically ✿✿✿✿✿✿✿✿✿✿✿✿✿ (Pequeno, 2014), which deducts to an average CH 4 emission of 0.05 nmol m −2 s −1 caused by mounds of this species alone.
Non-species specific mound densities are known to vary strongly between and within ecosystems (Ackerman (2006) However, since colony size can differ strongly between species, these ratios cannot be used to correctly upscale mound emissions to ecosystem scale. To our knowledge, only Bandeira and Torres (1985) (as given in Martius et al. (1996)) assessed the ratio between nest-building termite biomass vs total termite biomass, and estimated it to be ∼0.16. Considering the limited literature on this subject, we prefer not too further extrapolate our mound emission measurements.
Before measurement of the bag sample, sample lines were flushed with bag sample air. Air samples were dried by a Nafion dryer and by a column of magnesium perchlorate. Measurements were corrected for pressure and temperature variations as 485 well as for cross-sensitivities (Hammer et al., 2013). For more information on this instrument, please refer to Griffith et al. (2012). To determine the δ 13 C of the CO 2 emitted by the termite mounds, Keeling plots were used (Pataki et al., 2003).  (Quesada et al., 2010).

A2 Mound
Chamber CO concentrations ranged between 120 and 220 ppb ✿✿✿✿ nmol ✿✿✿✿✿✿ mol −1 , and showed a clear uptake on all days and 515 for all mounds, ranging between -0.04 to -0.78 nmol mound −1 s −1 (Fig. A1). The 'blank' soil location showed emissions between 0.31 and 0.52 nmol collar −1 s −1 . Termite mound uptake has been observed before by Khalil et al. (1990). We expect that the observed uptake is caused by aerobic CO-oxidising bacteria in the mound, which are also responsible for the CO uptake in (tropical) soils (Conrad, 1996;Kisselle et al., 2002;Liu et al., 2018;Potter et al., 1996;Whalen and Reeburgh, 2001;Yonemura et al., 2000a). Soil CO uptake is dependent on atmospheric CO and therefore often limited by low soil diffu-520 sivity (Sun et al., 2018;Yonemura et al., 2000b). The dry porous mound material (Martius et al., 1993) is therefore a suitable place for CO uptake.The observed emissions of the blank (soil) collar (0.31-0.52 nmol collar −1 s −1 ) are likely caused by the counteracting abiotic production, driven by temperature and radiation (King et al., 2012;Lee et al., 2012;Pihlatie et al., 2016; , or by a lesser studied anaerobic biological process (Moxley and Smith, 1998). While we expect that both soil uptake and emission are taking place in the blank soil collar (Kisselle et al., 2002;Liu et al., 2018;Potter et al., 1996;Van Asperen et al., 2015) 525 , it is likely that soil uptake is limited due to the low diffusivity of the wet valley soil, wherefore production becomes the dominant process.
Studies reporting values on mound emitted δ 13 C of have not been found. Based on our measurements, no significant difference in the δ 13 C between mound and soil emitted was found (-33.7 ‰ (se=2. representative for other moundsor soils in the valley, and to investigate whether an isotopic difference exists between mound and soil emitted ✿✿✿✿✿✿✿✿✿✿ soil-emitted CO 2 , more measurements would be needed.
Author contributions. HA designed and performed the field experiment, and wrote the paper, JA was responsible for the determination of the termite species, and gave input on the entomology part of the research, BF and AA provided access to the logistics and infrastructure of 550 the field site, JA, TW, BF, AA and JN reviewed and commented on the paper.
Competing interests. The authors declare that they have no conflict of interest.
(LBA)). We would also like to express our gratitude to the staff of LBA, for providing logistics, advice, and support during different phases of this research. In addition, we would like to thank Thiago de Lima Xavier and Leonardo Ramos de Oliveira for their advice in planning the technical parts of the experiment. Furthermore, we would like to acknowledge the group 'Department of Aquatic Biology and Limnology' Conrad, R.: Soil microorganisms as controllers of atmospheric trace gases (H2, CO,CH4,OCS,N2O,and NO)., Microbiological reviews, Zimmerman, P., Greenberg, J., Wandiga, S., and Crutzen, P.: Termites: a potentially large source of atmospheric methane, carbon dioxide, and molecular hydrogen, Science, pp. 563-565, 1982.          (Lepage and Darlington, 2000)); c) population estimate ✿✿✿ CSE ✿ based on mound surface area (given in Table 1), by use of mound termite surface values (5.6-16.7 termite cm −2 (Lepage and Darlington, 2000)); d) Population estimate ✿✿✿✿ CSE based on mound volume(given in Table 1), by species-specific volume-population equation of y=47.94*x 0.47 (x is mound volume (L), y is colony biomass (g)), as given by Pequeno et al. (2013) Overview of termite-derived and emissions, based on two different approaches. For comparison, the lowest row shows total (not termite-specific) ecosystem and flux values, measured at the same field site by previous studies. a) Querino et al. (2011) performed Eddy Covariance (EC) above-canopy flux measurements, and reported an averaged EC flux of ∼2 nmol m −2 s −1 ; b) Chambers et al. (2004) quantified different respiratory sources in this ecosystem, and estimated the total ecosystem respiration to be 7.8 µmol m −2 s −1 .