Michel Diouf
email: michel.diouf@u-pec.fr

David Sillam-Dussès

Vanessa Alphonse

Sophie Frechault

Edouard Miambi

Philippe Mora

Mercury species in the nests and bodies of soil-feeding termites, Silvestritermes spp. (Termitidae, Syntermitinae), in French Guiana

Keywords: soil-feeding termites, Syntermitinae, soil mercury, mercury methylation, bio-concentration

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mercury (Hg) is a pervasive neurotoxic heavy metal that has been introduced in diverse ecosystems through various mechanisms including natural erosion of the parent bedrock and anthropogenic activities (Science for Environment Policy, 2017). Living organisms are generally more tolerant of inorganic forms of Hg than its organic forms. Methylmercury (MeHg), a major form of organic mercury in food chain [START_REF] Benford | EFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific Opinion on the risk for public health related to the presence of mercury and methylmercury in food[END_REF][START_REF] Rice | Environmental mercury and its toxic effects[END_REF], is membrane-permeable, thiol reactive [START_REF] Clarkson | The toxicology of mercury and its chemical compounds[END_REF] and with a long biological half-life [START_REF] Jo | Estimation of the biological half-life of methylmercury using a population toxicokinetic model[END_REF]. It is therefore especially toxic and tends to accumulate in animal tissues, especially in longer-lived organisms and in predatory species [START_REF] Scheuhammer | Effects of environmental methylmercury on the health of wild birds, mammals, and fish[END_REF]). One of the main sources of MeHg in biota is the biotransformation of inorganic Hg to MeHg by microbial methylators carrying the hgcAB gene cluster that is required for Hg methylation in anoxic conditions [START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF][START_REF] Poulain | Cracking the mercury methylation Code[END_REF][START_REF] Gilmour | Mercury methylation by novel microorganisms from new environments[END_REF]). Aquatic microorganisms have been widely recognized as Hg biotransformers and, consequently, Hg methylation has usually been associated with aquatic food webs, sometimes extending to terrestrial invertebrates feeding on aquatic organisms [START_REF] Cristol | The movement of aquatic mercury through terrestrial food webs[END_REF]Becker et al. 2018;[START_REF] Bartrons | Taking the trophic bypass: Aquatic-terrestrial linkage reduces methylmercury in a terrestrial food web[END_REF][START_REF] Saxton | Maternal transfer of inorganic mercury and methylmercury in aquatic and terrestrial arthropods[END_REF]. There is now, however, a paradigm shift with increasing evidence of Hg biotransformation by various terrestrial invertebrates, amongst which earthworms [START_REF] Zhang | Bioaccumulation of total and methyl mercury in three earthworm species (Drawida sp., Allolobophora sp., and Limnodrilus sp.)[END_REF][START_REF] Rieder | Accumulation of mercury and methylmercury by mushrooms and earthworms from forest soils[END_REF][START_REF] Álvarez | Mercury methylation, uptake and bioaccumulation by the earthworm Lumbricus terrestris (Oligochaeta)[END_REF][START_REF] Álvarez | Using species-specific enriched stable isotopes to study the effect of fresh mercury inputs in soil-earthworm systems[END_REF][START_REF] Rieder | Accumulation of mercury and methylmercury by mushrooms and earthworms from forest soils[END_REF][START_REF] Álvarez | Mercury methylation, uptake and bioaccumulation by the earthworm Lumbricus terrestris (Oligochaeta)[END_REF][START_REF] Kaschak | Biotic methylation of mercury by intestinal and sulfate-reducing bacteria and their potential role in mercury accumulation in the tissue of the soil-living Eisenia foetida[END_REF]) and isopods [START_REF] Jereb | Transformations of mercury in the terrestrial isopod Porcellio scaber (Crustacea)[END_REF]. This extends the process beyond aquatic and wetland ecosystems (reviewed by [START_REF] Mahbub | Mercury toxicity to terrestrial biota[END_REF]).

Nevertheless, the potential for Hg methylation significant differ between invertebrates in general, and between arthropod taxa in particular as shown by Podar and colleagues [START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF] who found that only 4 metagenomes carried the hgcAB gene cluster out 27 metagenomes for arthropod microbiomes. Moreover, most studies demonstrating methylation capability in terrestrial invertebrates have used diets spiked with Hg. There are few studies addressing the effect of mercury contamination on terrestrial biota in natural conditions and even fewer for soil-inhabiting social insects which have high population densities in restricted habitats. In the invasive red fire ant (Solenopsis invicta), high levels of total mercury typical of aquatic insects have been reported, with even higher levels in reproductive males [START_REF] Helms | Invasive fire ants contain high levels of mercury[END_REF]. They only studied total Hg, but authors suggested that these high levels were due to predation on aquatic fauna with high levels of MeHg, rather than to environmental exposure to inorganic mercury. Besides ants, the production of MeHg by the wood-feeding Australian termite

Mastotermes darwiniensis has been reported from a feeding experiment with an artificial diet spiked with Hg and cultures of the gut-derived sulfate-reducing bacteria isolates showed Hg methylation activity [START_REF] Limper | Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis[END_REF]). We are not aware of any study on the effect of soil Hg on soil-living social insects that are primary consumers that are not only directly exposed to soil Hg, but also interact with Hg through the consumption and biotransformation of Hg-containing substrates.

Termites are fully social insects living in highly populated colonies. Besides the brood (eggs and larvae), termite colonies include various castes that are devoted to different tasks such as reproductives (kings, queen, nymphs), and sterile castes (workers and soldiers) [START_REF] Eggleton | An Introduction to Termites: Biology, Taxonomy and Functional Morphology[END_REF]. The foraging activities are accomplished only by the worker caste. The majority of termites are wood-feeders ie feed on woody materials at various levels of humification [START_REF] Donovan | The effect of a soil-feeding termite, Cubitermes fungifaber (Isoptera: Termitidae) on soil properties: Termites may be an important source of soil microhabitat heterogeneity in tropical forests[END_REF][START_REF] Eggleton | Feeding groups, lifetypes and the global ecology of termites[END_REF]. However, about one-third of known termite species are soil-feeders, feeding on mineral soil with little recognizable plant material [START_REF] Donovan | The effect of a soil-feeding termite, Cubitermes fungifaber (Isoptera: Termitidae) on soil properties: Termites may be an important source of soil microhabitat heterogeneity in tropical forests[END_REF][START_REF] Inward | A comprehensive phylogenetic analysis of termites (Isoptera) illuminates key aspects of their evolutionary biology[END_REF]). Nests of these soil-feeding termites are built by workers using firm amalgams of soil from the 10 top cm and feces. The enrichment in organic matter and in small-size particles such as clay and limon relative to the soil from the nearby environment is one of the salient features of the nest materials of soil-feeding termites. These are particularly abundant and diverse in tropical rain forests, appearing as one of the main contributors in soil processing and humification. Through their soil consumption and burrowing activities, they have a major effect on soil physical, chemical and biological properties, which, in turn, affects the cycling and speciation of elements, including trace metals, in the soil [START_REF] Brauman | Effect of gut transit and mound deposit on soil organic matter transformations in the soil feeding termite: A review[END_REF][START_REF] Donovan | The effect of a soil-feeding termite, Cubitermes fungifaber (Isoptera: Termitidae) on soil properties: Termites may be an important source of soil microhabitat heterogeneity in tropical forests[END_REF][START_REF] Sarcinelli | Chemical, physical and micromorphological properties of termite mounds and adjacent soils along a toposequence in Zona da Mata, Minas Gerais State, Brazil[END_REF][START_REF] Sarcinelli | Soil modification by termites in a sandy-soil vegetation in the Brazilian Atlantic rain forest[END_REF][START_REF] De Lima | Soil physical and chemical properties in epigeal termite mounds in pastures[END_REF]. Their effect on soil properties could be compared to that of earthworms whose effects on heavy metals in soil have been more widely studied. However, soil-feeding termites are ecologically distinct from earthworms in various ways: unlike earthworms, they are social and thus live in densely populated colonies, they have longer-lasting biostructures than worm casts and burrows and they have a more complex gut structure hosting a more complex microbiota.

Soil-feeding termites are the dominant feeding group of termites in various Amazonian rainforest ecosystems. In various oxisols of these ecosystems the soil mercury concentration is between 0.25 and 0.5µg.g -1 [START_REF] Lechler | Elevated mercury concentrations in soils, sediments, water, and fish of the Madeira River basin, Brazilian Amazon: A function of natural enrichments?[END_REF][START_REF] Richard | Mercury levels of soils, sediments and fish in French Guiana, South America[END_REF], well above the normal levels in soil (0.05 to 0.08 µg.g -1 ) reported by the World Bank (1999). They also exceed the limits relative to soil organic matter (SOM) (0.5 µg.g -1 SOM) proposed by de Vries and collegues (de Vries et al. 2007) and relative to the soil (0.13 µg.g -1 of dry weight soil) proposed by Tipping and colleagues [START_REF] Tipping | Critical Limits for Hg(II) in soils, derived from chronic toxicity data[END_REF]. The topsoil of these ecosystems is therefore subject to a generalized, chronic excess of Hg, although the background mercury level (in the bedrock) is significantly lower (0.020 -0.1 µg.g -1 ) [START_REF] Aula | Levels of surrounding, mercury in the Tucurui reservoir and itsarea in Para, Brazil[END_REF][START_REF] Pfeiffer | Environmental fate of mercury from gold mining in the Brazilian Amazon[END_REF]. This Hg overload is believed to arise mainly from atmospheric Hg deposition over several millions of years, which has been particularly high from the end of the 15 th century [START_REF] Roulet | Le mercure dans les sols d'Amazonie : origine et comportement du mercure dans les couvertures ferrallitiques du bassin amazonien et des Guyanes[END_REF][START_REF] Obrist | A review of global environmental mercury processes in response to human and natural perturbations: Changes of emissions, climate, and land use[END_REF]. In forest soils on the French Guiana plateau, there is a high abundance and diversity of soil-feeding termites, including many endemic species, which may indicate that this chronic exposition to these high levels of mercury may not be directly harmful to these termites. Some animals can thrive at contamination levels generally considered to be lethal by sequestrating and accumulating the toxic agent in non-essential tissues [START_REF] Lanno | The bioavailability of chemicals in soil for earthworms[END_REF][START_REF] Mahbub | Mercury toxicity to terrestrial biota[END_REF]). Nevertheless, they may still pose a risk for the biota by exposing their predators and the upper levels of the food chain to high levels of mercury in their food. For assessing this risk, Hg storage in animal tissues is more relevant than the sensitivity to mercury exposure.

Our study addresses the processing of soil mercury by soil-feeding termites from the genus Silvestritermes. We compared the concentrations of various mercury species in the soil around the nest harvested by the termites and the concentrations in their nests. Silvestritermes is an abundant genus of soil-feeding termites in the neotropical forests, belonging to the subfamily Syntermitinae (Termitidae family) that is endemic to this region. As for organic mercury, we compared concentrations of methylmercury in the nest, termite bodies and termite body parts to those of the nearby soil. We discuss the concerns raised by Hg biotransformation by termites considering the role of termitophagy in the food webs.

Material and Methods

Sampling and sample processing

The main sampling site was at Crique Combat, which is an area of 1 km 2 covered by a primary rainforest, near the village of Cacao (Figure S1, Supplementary information). The soil in this area is well-characterized since this site has hosted several research programs (such as "Mercury in Guyana" (2002)(2003)(2004)(2005)(2006)(2007) run by CNRS (French National Research Organization)) and several thesis projects [START_REF] Guedron | Mercury speciation in a tropical soil association; Consequence of gold mining on Hg distribution in French Guiana[END_REF][START_REF] Harris-Hellal | Mercury mobilization by chemical and microbial iron oxide reduction in soils of French Guyana[END_REF]. Six colonies of the soil-feeding termite Silvestritermes minutus and four colonies of S. holmgreni were randomly sampled from the oxisol (USDA Soil Taxonomy) at the top of the slope, with a minimum distance of 20 m between them. Both species build well-defined nests that are spherical to ellipsoid structures emerging a few centimeters above the ground level. For S. minutus, nests were generally built on plants sprouts, thus penetrated and consolidated by a network of plants roots for mature colonies (see Fig. S2). As for S. holmgreni, their nests are generally built laterally against the basis of tree trunks.

Nests were partially broken to collect a representative selection of the nest material, with termite individuals inside. A composite soil sample was collected from nearby each nest. This was a mix of four cores from the 0-10 cm layer where soil-feeding termites are mainly found [START_REF] Brauman | Effect of gut transit and mound deposit on soil organic matter transformations in the soil feeding termite: A review[END_REF], at a radius of 2 m from the nest. Then, the termites were separated from the nest material using plastic tweezers. Only workers were collected for mercury analyses: these are the only caste feeding directly on the soil, unlike the other castes which are fed by workers. The nest material and the soil were hand sorted to remove roots and pebbles and then crushed. All samples (workers, nest material and soil) were stored in sterile, trace metal-free Falcon tubes and frozen at -20°C. Then, they were transported in dry ice to mainland France, where they were frozen at -80°C until analysis.

Termites from the second sampling site near Petit Saut (S. minutus only) were used to analyze the MeHg distribution in their bodies. Whole colonies were collected by disrupting at the base of nest the roots that maintain them tied to the ground (see fig. S2B). They were shipped to mainland France. Nearby soil samples were collected and transported as described above. Samples were processed within two days of arrival at the laboratory. The guts were removed from the workers using fine sterile tweezers and the gut-free carcasses, with the head and the legs, were kept as separate samples. The guts and gut-free carcasses for each colony were pooled in separate sterile microtubes. All the samples (soil, guts and gut-free carcasses) were stored at -80°C until analysis.

Five to 10 soldiers were collected from each colony for the identification of the termite species. The species identity was checked from morphological criterion and by DNA barcoding, based on the cytochrome oxidase I gene. For DNA extraction from the head of soldiers, PCR amplification and sequencing, the method used by Fougeyrollas and colleagues was applied [START_REF] Fougeyrollas | Dispersal and mating strategies in two neotropical soil-feeding termites, Embiratermes neotenicus and Silvestritermes minutus (Termitidae, Syntermitinae)[END_REF].

Total mercury and mobile mercury analyses of the nest material and the nearby soil

Total mercury (THg) was determined using an atomic absorption spectrometer AMA-254 (Advanced Mercury Analyser, Altec, Ltd., Czech Republic) following the protocol described by [START_REF] Sysalová | Determination of the total mercury in contaminated soils by direct solid sampling atomic absorption spectrometry using an AMA-254 device and radiochemical neutron activation analysis[END_REF]. This measures trace levels mercury in samples directly without pre-treatment by mercury extraction. One hundred milligrams of soil or nest material were placed in a nickel boat and introduced into the spectrometer. After drying at 120°C, the samples were combusted at 550°C for 150 sec in an oxygen-saturated atmosphere transforming the total mercury into elementary mercury (Hg 0 ) which is readily adsorbed onto a gold-trap. The mercury was then released by heating the gold-trap to 450°C and quantified by atomic adsorption spectrometry at a wavelength of 253.7 nm.

The mobile, toxic mercury (MHg) fraction, comprising soluble inorganic Hg and alkyl-Hg forms (methyl and ethyl Hg), was extracted using acidic ethanol with 1:1 (v/v) 2% hydrochloric acid and 10% ethanol [START_REF] Han | Speciation of mercury in soil and sediment by selective solvent and acid extraction[END_REF][START_REF] Reis | Extractability and mobility of mercury from agricultural soils surrounding industrial and mining contaminated areas[END_REF][START_REF] Frentiu | Determination, speciation and distribution of mercury in soil in the surroundings of a former chlor-alkali plant: assessment of sequential extraction procedure and analytical technique[END_REF]. Except for the initial sample weight (500 mg in this study), the mercury was extracted using the protocol described by [START_REF] Frentiu | Determination, speciation and distribution of mercury in soil in the surroundings of a former chlor-alkali plant: assessment of sequential extraction procedure and analytical technique[END_REF]. All samples were then filtered through PTFE filters (0.45 µm) and acidified to a pH of 1. This mercury fraction was measured using the AMA-254 as described above for the solid samples, manually injecting 200 µl of the filtrate.

The detection limit ranged from 0.01 to 500 ng of Hg in a sample. Dilutions from a 1000 mg.l -1 mercury standard solution (Hg PlasmaCAL, SCP Science) were used for calibration. The data acquisition was driven by AMA 254 V5.0.2.4 software . The routine quality assurance metrics included in the standardized analytical method were fulfilled by all samples. The mercury content of the standard reference materials namely LGC6139 (River clay sediment-Metals) and BCR 277R (Estuarine sediment) was recovered within certified limits:1.2 (±0.05) µg.g -1 and 0.128 (±0.017) µg g -1 respectively. Likewise, triplicated samples passed the method criteria of <20% RSD (Relative Standard Deviation) for liquids samples and <5 % RSD for solids samples.

Analysis of methylmercury in termites, nests and soil samples

MeHg concentrations were measured at the Wisconsin State Laboratory of Hygiene (WSLH), by the Inorganic Chemistry Unit of the Environmental Health Division. After extraction by grinding freeze-dried samples with an agate mortar and pestle and digesting known amounts of matter with KOH-methanol, MeHg was measured by aqueous ethylation, purge and trap, desorption and Cold Vapor Atomic Fluorescence Spectrophotometer (CVAFS, Brooks Rand model III) following a standard procedure (ESS INO METHOD 545.1 rev. 4). All samples passed the routine assurance quality metrics included in this standardized analytical method. For example, both NRCC standard materials (National Research Council Canada), TORT-3 and DORM-3 were recovered within the certified limits 105% and 119% respectively. Also, matrix spikes on samples were recovered at 108% and 95.4%. Finally, duplicated samples passed the acceptance criteria of <35% RSD. On a dry weight basis, duplicate 1 was 481 and 383 ng.g -1 and duplicated 2 was 2.08 and 2.83 ng.g -1 dry weight.

Statistical analyses

The statistical analyses were performed using the Statgraphics software (Centurion XVI, Sigma Plus, France). For the concentrations of total (THg), mobile Hg (MHg) and the ratio of MHg to THg, the data was not normally distributed (Shapiro-Wilk test at 95% confidence). Therefore, the non-parametric Kolmogorov-Smirnov test was used to assess the significance of the differences in concentration, with p < 0.05. MeHg concentrations were, however, normally distributed.

Differences between samples of each termite species were analyzed using one-way ANOVA. Correlations between the various mercury fractions were tested using Spearman's rank correlation (p < 0.05) and the correlation curves were constructed using ggplot2 and ggpubr packages in R version 3.4.4.

Results

Concentration of total (THg) and mobile Hg (MHg) in S. minutus nests and the nearby soil

THg concentration was not statistically different between nests and the nearby soil (p=0.149) (fig. 1A). The THg concentration in the nests appear to be related to the concentration in the nearby soil as shown by the high positive correlation between THg concentrations in the two compartments (R=0.93; p=0.007 (fig. 2A).

The MHg concentration in the nest material was significantly lower than in the soil (fig. 1B). The MHg concentration was less variable between the nest materials than between the soils. Unlike THg, the MHg concentrations in the nest materials were not correlated with that in the nearby soil. However, the MHg concentration in both the nest and the soil seems to be related to THg concentration (R=0.76; p=0.0041) (fig. 2B). The mobile mercury percentage (relative to THg) was significantly lower in the nest than in the soil (p=0.037) (fig. 1C) and was correlated with the MHg concentration (R=0.85; p=0.0005) rather than with the THg concentration (R=0.38; p=0.22) (fig. 2 C &D).

MeHg concentration in soil, nests and the termite whole bodies of S. minutus and S. holmgreni

At the Crique Combat site, the concentration of MeHg was lower in the nearby soil than in all other samples of S. minutus, covering only a small fraction of the THg (0.18 ± 0.10 %) (Table 1). For this termite species, unlike THg and MHg, the MeHg concentrations in the nest material were far higher than in the nearby soil and covered a significantly higher fraction of THg (4.73 ± 4.42%). MeHg concentration was independent of THg or MHg concentrations according to the correlation tests. The bodies of S. minutus had higher concentrations of MeHg than the nearby soil and nests. Since termite workers reside either in the soil (for the foraging tasks) or in the nest (for the social and building tasks), the bio-concentration factor (BCF) of MeHg in the termite body was calculated relative to both habitats. The resulting BFC was much higher relative to the nearby soil (57.84 ± 25.85), but remained substantial high relative to the nest (3.76 ± 1.61).

While the body concentration of MeHg was uncorrelated with the concentration in the soil, it was significantly correlated positively with the concentration in the nest (R=0.89).

For the congeneric species, S. holmgreni, living in sympatry with S. minutus, except the lack of statistical difference between MeHg in the soil and in the nest, the results were similar, with even a higher BCF in the bodies relative to the nest and to the soil.

The second set of samples, from Petit Saut, was analyzed to determine whether the high amounts of MeHg in the body of termites were restricted to the gut or widespread through the tissues. The MeHg concentration was measured for the whole gut (accounting for over two-thirds of the termite body weight) and for the gut-free carcasses separately, as well as for the nest and the nearby soil (Table 2). The MeHg concentration of the nearby soil was below the detection limit of the analysis method while in the nests, it was in the same order of magnitude as in nests from Cacao. The MeHg concentration in the termite body parts was much higher than in the soil and nests. The highest concentrations of MeHg were found in the termite carcasses. The BCF in the body parts could not be calculated relative to the soil, but was very high relative to the nest material both for the termite guts and for the gut-free carcasses.

Discussion

In this study, we assess various mercury forms in nests and the bodies of soil-feeding termites. Several studies have pointed out the effects of termite activities on soil physical and chemical properties and, thereby, on soil heterogeneity at the landscape level [START_REF] Jouquet | Influence of termites on ecosystem functioning. Ecosystem services provided by termites[END_REF]). These effects on soil properties are characteristic of all termite feeding groups, even including the majority of termites feeding on above ground litter and wood material. However, soil-feeding termites consume, partially or exclusively, soil [START_REF] Inward | A comprehensive phylogenetic analysis of termites (Isoptera) illuminates key aspects of their evolutionary biology[END_REF][START_REF] Eggleton | Feeding groups, lifetypes and the global ecology of termites[END_REF] in a similar way to earthworms, and have a more significant contribution [START_REF] Brauman | Effect of gut transit and mound deposit on soil organic matter transformations in the soil feeding termite: A review[END_REF][START_REF] Donovan | The effect of a soil-feeding termite, Cubitermes fungifaber (Isoptera: Termitidae) on soil properties: Termites may be an important source of soil microhabitat heterogeneity in tropical forests[END_REF][START_REF] Sarcinelli | Chemical, physical and micromorphological properties of termite mounds and adjacent soils along a toposequence in Zona da Mata, Minas Gerais State, Brazil[END_REF][START_REF] Sarcinelli | Soil modification by termites in a sandy-soil vegetation in the Brazilian Atlantic rain forest[END_REF][START_REF] De Lima | Soil physical and chemical properties in epigeal termite mounds in pastures[END_REF]. This is particularly true in neotropical rainforests where they are among the most representative feeding-groups of termites in various forest habitats [START_REF] Ackerman | Termite (Insecta: Isoptera) species composition in a primary rain forest and agroforests in Central Amazonia[END_REF][START_REF] De Sales Dambros | Road-associated edge effects in Amazonia change termite community composition by modifying environmental conditions[END_REF][START_REF] Souza | Effects of habitat fragmentation on Amazonian termite communities[END_REF][START_REF] Davies | Environmental and spatial influences upon species composition of a termite assemblage across neotropical forest islands[END_REF]. This is the case in French Guiana where up to 73 species of termites are soil-feeding out of 100 species locally recorded [START_REF] Davies | Environmental and spatial influences upon species composition of a termite assemblage across neotropical forest islands[END_REF].

The THg concentration in the soil at Crique Combat averaged 0.383 µg.g -1 of dry soil, which is within the range of concentrations recorded in various Amazonian oxisols (0.250-0.500 µg.g -1 ) [START_REF] Lechler | Elevated mercury concentrations in soils, sediments, water, and fish of the Madeira River basin, Brazilian Amazon: A function of natural enrichments?[END_REF]Richardet al. 2000) and, in particular, previously recorded for oxisols from the same study site [START_REF] Guedron | Mercury speciation in a tropical soil association; Consequence of gold mining on Hg distribution in French Guiana[END_REF][START_REF] Silva | Toxicity of mercury to the earthworm Pontoscolex corethrurus in a tropical soil of French Guiana[END_REF]. This THg concentration is well above normal levels in soil (0.05 to 0.08 µg.g -1 ) reported by the World Bank (1999). It also exceeds proposed limits relative to SOM (0.5 µg.g -1 SOM, de Vries et al. 2007), as the SOM in this horizon is around 5.6 ± 0.6% dry soil (Da [START_REF] De Sales Dambros | Road-associated edge effects in Amazonia change termite community composition by modifying environmental conditions[END_REF], and relative to the soil (0.13 µg.g -1 dry soil, [START_REF] Tipping | Critical Limits for Hg(II) in soils, derived from chronic toxicity data[END_REF]. Soil-feeding behavior in this case may result in a handling of huge amounts of mercury by termites. Indeed, a humid savannah ecosystem, soil-feeding termites from the genus Cubitermes were reported to ingest annually up to 4.5 kg m -2 [START_REF] Lavelle | Soil function in a changing world: the role of invertebrate ecosystem engineers[END_REF].

Likewise, a daily consumption of soil ranging from 0.72 to 0.911 mg of soil has been reported for another species Cubitermes [START_REF] Okwakol | Estimation of soil and organic matter consumption by termites of the genus Cubitermes[END_REF], that is almost the equivalent of its own weight (≈10 mg). Though to our knowledge, the daily consumption of soil was not yet reported for the Silvestritermes species understudy, the fate of the Hg that is ingested all the time with soil emerged as a crucial question.

The THg concentration for each nest was below that of the nearby soil, although this was not statistically significant, but there was a significant positive correlation between THg concentrations in the soil and in the nest material, indicating that the THg concentration in the nests of soil-feeding termites could be predicted from the THg concentration in the soil.

The toxicity to biota of heavy metals in general, and of mercury in particular, depends on the bioavailable soluble fraction rather than on the total concentration.

Extracting Hg with an acid ethanol solution can be used to assess the mobile, toxic fraction comprising of soluble inorganic and alkyl Hg species [START_REF] Han | Speciation of mercury in soil and sediment by selective solvent and acid extraction[END_REF]. We anticipated that soil processing by the termites should release adsorbed mercury, and therefore, lead to a higher fraction of mobile mercury in the nest than in the soil. Unexpectedly, the MHg concentration and its proportion of THg were significantly lower in the nests than in the nearby soil. Soil processing did not therefore increase the MHg concentration, unless the mobilized Hg was then converted into volatile or immobile organic forms.

As far as we know, no data are available on the effect of termites' nest building activities on the mercury naturally present in the soil. As stated above, nests of soil-feeding termites are a fine mixture of soil and fecal matter and, as such, are not strictly comparable with true feces of other arthropods. Various factors related either to the foraging and building behavior or to mercury biotransformation after ingestion could explain this decrease in the nests, especially for the MHg fraction. The first possibility is that soil-feeding termites preferentially select soil fractions with less affinity for Hg during harvesting. The question of food particle selection by soil-feeding termites remains unanswered [START_REF] Brauman | Effect of gut transit and mound deposit on soil organic matter transformations in the soil feeding termite: A review[END_REF][START_REF] Brauman | Soil-Feeding Termites: Biology, Microbial Associations and Digestive Mechanisms[END_REF], but their nests are commonly enriched in organic matter (OM) and in small-size particles such as clay [START_REF] Brauman | Effect of gut transit and mound deposit on soil organic matter transformations in the soil feeding termite: A review[END_REF][START_REF] Brauman | Soil-Feeding Termites: Biology, Microbial Associations and Digestive Mechanisms[END_REF][START_REF] Fall | Comparative distribution of organic matter in particle and aggregate size fractions in the mounds of termites with different feeding habits in Senegal: Cubitermes niokoloensis and Macrotermes bellicosus[END_REF][START_REF] Sarcinelli | Chemical, physical and micromorphological properties of termite mounds and adjacent soils along a toposequence in Zona da Mata, Minas Gerais State, Brazil[END_REF][START_REF] Sarcinelli | Soil modification by termites in a sandy-soil vegetation in the Brazilian Atlantic rain forest[END_REF][START_REF] Ngugi | Nitrogen mineralization, denitrification, and nitrate ammonification by soil-feeding termites: A15N-based approach[END_REF][START_REF] De Lima | Soil physical and chemical properties in epigeal termite mounds in pastures[END_REF]. Strangely, both SOM and clay are considered to have high affinity for, and are often positively correlated with, soil Hg [START_REF] Gabriel | Principal biogeochemical factors affecting the speciation and transport of mercury through the terrestrial environment[END_REF][START_REF] Różański | Bioavailability and mobility of mercury in selected soil profiles[END_REF]. Particle selection does not, therefore, seem to explain the low mercury concentration in the nests. The lower concentration of THg and, especially, MHg in the nests could be related to the microbial transformation of soil Hg. From ingestion by the termites, up to deposition on the lining of nest, the soil moves through various oxic and redox conditions [START_REF] Brune | The termite gut microflora as an oxygen sink: microelectrode determination of oxygen and pH gradients in guts of lower and higher termites[END_REF][START_REF] Brune | Microecology of the termite gut: structure and function on a microscale[END_REF][START_REF] Kappler | Dynamics of redox potential and changes in redox state of iron and humic acids during gut passage in soil-feeding termites (Cubitermes spp.)[END_REF] compatible with the microbial transformation of Hg(II), either by dissimilatory reduction into elemental Hg (Hg 0 ) or by methylation into MeHg. The reduction of Hg(II) into volatile Hg 0 is performed by bacteria carrying the mer operon is the most common Hg dissimilatory pathway [START_REF] Boyd | The mercury resistance operon: From an origin in a geothermal environment to an efficient detoxification machine[END_REF]. It operates under aerobic conditions that are found in various gut sections (fore-gut, mid-gut and the terminal subsections of the hind-gut) and even at the periphery of the paunch that is believed to be internally anaerobic [START_REF] Brune | Microecology of the termite gut: structure and function on a microscale[END_REF]. Moreover, it is possible that Hg is reduced under anaerobic conditions through constitutive pathways whose mechanisms are less well documented [START_REF] Wiatrowski | Novel reduction of mercury(ii) by mercury-sensitive dissimilatory metal reducing bacteria[END_REF]. Anoxic conditions are found mainly in the paunch that is the largest part of the termites' gut. To the best of our knowledge, this process has not yet been explored in termites. Although the conditions in the gut are potentially favorable to Hg reduction and volatilization, its occurrence should be substantiated by measuring mercury fluxes and testing for the presence of the genes or microorganisms involved.

Methylation of Hg to MeHg was the other possible pathway explaining the depletion of inorganic Hg in the nests since the MeHg concentration increased as MHg and THg decreased, MeHg concentrations being on average 17 times higher in the nests than in the soil. This enrichment could hardly be explained by the bioconcentration of the small amounts of MeHg from the ingested soil. Additionally, part of the ingested MeHg might have been assimilated or demethylated during the gut transit [START_REF] Nolde | Reduction and methylation of mercury in the terrestrial isopod Porcellio scaber (Crustacea) and its environment[END_REF]. This enrichment is probably, therefore, the result of de novo synthesis of MeHg through Hg-methylation by the termites' microbiota during soil processing. This hypothesis is supported by the MeHg concentration in the termites which was several orders of magnitude greater than in the soil or nests. The MeHg concentrations in the body were similar for S. minutus and the congeneric species S. holmgreni but the latter had even higher average BCFs values relative to the soil and relative to the nests suggesting that the high MeHg enrichment in termites could be a widespread phenomenon among Guyanese soil-feeding termites. The mercury methylation potential of termites was first reported for Mastotermes darwiniensis, a wood-feeding species endemic to Northern Australia [START_REF] Limper | Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis[END_REF]. Unlike soil-feeding termites with purely prokaryotic microbiotas, M. darwiniensis also hosts protozoa (like all lower termites), and is the sole extant member of the Mastotermitidae family, the most basal in the termite phylogeny. As well as the differences between termite taxa, the experimental approach was different. The Hg-methylation potential of M. darwiniensis was tested in a short-term experiment (14 days) based on an artificial diet (sawdust) spiked with much higher Hg concentrations (25-250 µg.g -1 HgCl2) than in the soil of our study. The resulting MeHg concentrations in the termites were in the same range as in our study (15.9-53.8 ng.g -1 DW). Although the biological traits of the taxa may be different, the similar enrichment in MeHg in the body with much lower mercury concentrations under natural conditions may suggest that the length of exposure may significantly affect termites' accumulation of mercury in their bodies. Soil-feeding termites are also known to re-ingest fresh nest material [START_REF] Brauman | Soil-Feeding Termites: Biology, Microbial Associations and Digestive Mechanisms[END_REF], which could contribute to this body accumulation of MeHg, given its higher concentrations in the nest relative to the soil.

MeHg is the most problematic form of mercury for living organisms. Due to its affinity with thiol groups (R-SH) and selenol groups (R-SeH, that easily replace thiol groups in amino-acids), MeHg is readily binds to cysteine, tripeptide glutathione and cysteine moieties of proteins and enzymes (Wang et al. 2012) that are structurally and functionally pivotal in biological systems. As a result, MeHg is readily adsorbed into animal tissues and bioaccumulated throughout food chains [START_REF] Gaur | Biological influences of mercury on living organisms[END_REF]. The distribution of MeHg in the termite body was assessed to establish whether MeHg was limited to the gut content, and, therefore, likely to be transitory, or diffusely and durably stored within termite tissues. The MeHg concentrations in the guts and gut-free carcasses from Petit Saut were much higher than those in the soil and nests, in agreement with the higher concentration in the whole workers from Crique Combat. However, both body parts contained substantially higher MeHg levels than the whole workers from Crique Combat. MeHg concentrations were not directly measured in the whole body, but the higher concentrations in both body parts clearly indicate that workers from Petit Saut had higher whole body concentrations. The inorganic forms of mercury were not measured in Petit Saut. However, the MeHg concentrations in the nests, which, for Crique Combat, were closely correlated with the MeHg concentration in the body, was of the same order of magnitude as for nests from Crique Combat and soil MeHg concentrations were even lower than in soils from Crique Combat. More in-depth investigation is therefore needed to explain this difference between sites in MeHg concentrations in the termite bodies.

The highest average concentrations of MeHg were found in worker gut-free carcasses, rather than in the gut, but the difference was not statistically significant.

Moreover, the gut samples were mixtures of the food bolus and the gut-wall tissues, and as the MeHg concentration was lower in the nest which is (at least partly) constructed with feces, MeHg in the gut-wall tissue might have been underestimated. The main conclusion from the high MeHg concentration in the carcasses was that MeHg was not confined to the gut bolus and wall, but distributed through the termite tissues. Gut-free carcasses were also mixtures: integuments (cuticle), heads and legs which are more sclerotized body parts. In the Isopod Porcellio scaber, a higher proportion of MeHg (relative to the total MeHg or THg) was recorded in the residue and attributed to the high affinity of MeHg for the sylfhydryl (-SH) groups of some compounds involved in cuticle sclerotization [START_REF] Bittner | When quinones meet amino acids: Chemical, physical and biological consequences[END_REF]. The high proportion of sclerotized parts (head and legs) in these samples might explain the association of MeHg with the carcasses in our study.

Finer analyses, separating the gut wall from the content, and separating the various parts of the carcass, are, therefore, needed to shed light on the contribution of each part.

There is a growing body of data for THg concentrations for various arthropods under diverse conditions of mercury pollution but there is less data about the concentration of organic forms of mercury in terrestrial arthropods, and much less in social insects. The concentrations of MeHg measured here in termite tissues are much higher than those of THg reported for various ants, including invasive fire ants (Solenopsis invicta) for which the concentrations were considered to be significantly high [START_REF] Helms | Invasive fire ants contain high levels of mercury[END_REF]. The lack of information about the THg concentrations in the ants' habitat limits any comparison. MeHg concentrations in the bodies of various terrestrial arthropods at the same trophic level (primary consumers) have often been lower than those reported here for termites. This is the case for herbivorous insects (Locusta migratoria manilensis, Acrida chinensis) (1-12 ng.g-1 ) [START_REF] Zheng | Bioaccumulation of total and methyl mercury by Arthropods[END_REF], for pill bugs and ground beetles [START_REF] Ortiz | Total and monomethyl mercury in terrestrial arthropods from the central california coast[END_REF] in Hg-polluted environments. However, the MeHg concentrations within the range we found in termites was reported for the sapfeeding cicada, Cryptotympana atrata, in a polluted environment, probably due to a higher degree of exposure related to the longer lifespan and to a long underground larval stage (4-5 years) during which the larvae feed on the Hg-rich sap from roots [START_REF] Zheng | Total and methyl mercury contents and distribution characteristics in cicada, Cryptotympana atrata (Fabricius). Bulletin of S. minutus S. holmgreni MeHg (ng g -1 ) (Mean ± SD) Range (ng g -1 ) % MeHg (% of THg) MeHg (ng g -1 ) (Mean ± SD) Range (ng g -1 ) Nearby soils 0.66 ± 0.30 A 0[END_REF]. MeHg concentrations similar to or higher than those measured here in termites are fairly common in aquatic or wetland arthropods [START_REF] Ackerman | Invertebrate mercury bioaccumulation in permanent, seasonal, and flooded rice wetlands within California's Central Valley[END_REF][START_REF] Bates | Concentrations of methylmercury in invertebrates from wetlands of the Prairie Pothole Region of North America[END_REF] due to high Hg-methylation in these environments, as well as in terrestrial predators such as spiders, Odonata, centipedes and scorpions [START_REF] Bartrons | Taking the trophic bypass: Aquatic-terrestrial linkage reduces methylmercury in a terrestrial food web[END_REF][START_REF] Bates | Concentrations of methylmercury in invertebrates from wetlands of the Prairie Pothole Region of North America[END_REF][START_REF] Ortiz | Total and monomethyl mercury in terrestrial arthropods from the central california coast[END_REF][START_REF] Tavshunsky | Accumulation of methylmercury in invertebrates and masked shrews (Sorex cinereus) at an upland Forest-Peatland interface in Northern Minnesota, USA[END_REF][START_REF] Tsui | Variation in terrestrial and aquatic sources of methylmercury in stream predators as revealed by stable mercury isotopes[END_REF].

As well as the high MeHg concentration in the termites reported here, there is a large body of evidence supporting the hypothesis that termites can methylate the Hg naturally present in ingested food. The first argument is that sulfate reducing bacteria (SRB), which are the best-known group of Hg-methylators, are widespread in termite microbiotas from wood-feeding lower termites [START_REF] Trinkerl | Desulfovibrio termitidis sp. nov., a carbohydrate-degrading sulfate-reducing bacterium from the hindgut of a termite[END_REF][START_REF] Kuhnigk | A feasible role of sulfate-reducing bacteria in the termite gut[END_REF][START_REF] Fröhlich | Isolation of Desulfovibrio intestinalis sp. nov. from the hindgut of the lower termite Mastotermes darwiniensis[END_REF][START_REF] Dröge | In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata[END_REF][START_REF] Limper | Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis[END_REF] to higher termites (protozoa-free) including soil-feeders [START_REF] Brauman | Characterization of two sulfate-reducing bacteria from the gut of the soil-feeding termite, Cubitermes speciosus[END_REF][START_REF] Kuhnigk | A feasible role of sulfate-reducing bacteria in the termite gut[END_REF]. SRB from the genus Desulfovibrio, about 26% of known bacterial methylators [START_REF] Gilmour | Robust mercury methylation across diverse methanogenic Archaea[END_REF], and those from the genus Desulfarculus, are highly conserved across termite microbiotas (Abdul [START_REF] Rahman | A molecular survey of Australian and North American termite genera indicates that vertical inheritance is the primary force shaping termite gut microbiomes[END_REF], including in soil-feeders [START_REF] Brauman | Characterization of two sulfate-reducing bacteria from the gut of the soil-feeding termite, Cubitermes speciosus[END_REF][START_REF] Kuhnigk | A feasible role of sulfate-reducing bacteria in the termite gut[END_REF]. Additionally, several SRB strains isolated from termite guts can methylate Hg(II) in vitro [START_REF] Kaschak | Biotic methylation of mercury by intestinal and sulfate-reducing bacteria and their potential role in mercury accumulation in the tissue of the soil-living Eisenia foetida[END_REF][START_REF] Limper | Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis[END_REF], including a strain from a soil-feeding termite Cubitermes sp. [START_REF] Kaschak | Biotic methylation of mercury by intestinal and sulfate-reducing bacteria and their potential role in mercury accumulation in the tissue of the soil-living Eisenia foetida[END_REF]. The second argument is that the hgcAB gene cluster for Hg-methylation [START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF][START_REF] Poulain | Cracking the mercury methylation Code[END_REF] has been found in various higher termite metagenomes, including those of soil-feeding species [START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF][START_REF] Martín-Doimeadios | Is gastrointestinal microbiota relevant for endogenous mercury methylation in terrestrial animals?[END_REF]. Methanogenic archaea, including Hg methylators [START_REF] Gilmour | Robust mercury methylation across diverse methanogenic Archaea[END_REF], are also very prevalent in termite microbiotas (reviewed by [START_REF] Brune | Methanogens in the digestive tract of termites[END_REF] including those of soil-feeders [START_REF] Ohkuma | Phylogenetic relationships of symbiotic methanogens in diverse termites[END_REF][START_REF] Donovan | Comparison of Euryarchaea strains in the guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types[END_REF]). Since the bacterial and methanogenic archaeal methylators probably share the same micro-environment, the syntrophic interactions for Hg-methylation that are probably widespread in complex systems [START_REF] Yu | Syntrophic pathways for microbial mercury methylation[END_REF]) may also occur in the termite gut. All this evidence that termites are potentially Hg methylators, strongly suggest that the high MeHg concentrations in the termite bodies arise from the activity of Hg-methylators in the gut microbiota. Additional experiments comparing the extent and diversity of the hgcAB gene cluster in the gut, as a predictor of Hg-methylation potential, with the MeHg concentrations in the body tissues under various levels of mercury pollution are needed for a better understanding of the role of these microbial methylators.

Conclusion

To our knowledge, this is the first study addressing the effect of termites on the fate of soil mercury under natural conditions. Nests of the soil-feeding termites that significantly contribute to the heterogeneity of soils in tropical forests are depleted in inorganic mercury while the environmental levels of mercury were rather high. The turnover of their nests (erosion, colony death) reduces soil mercury concentrations at local scales. Organic mercury shows the opposite pattern with the nests and their inhabiting termites being hotspots of MeHg. These high MeHg concentrations found, especially in termite tissues, can be a concern, especially in heavily contaminated sites. This preoccupation is further justified by the findings that MeHg enrichment seems to be widespread within soil-feeding termites that are among the most common termites in neotropical rainforest ecosystems [START_REF] Ackerman | Termite (Insecta: Isoptera) species composition in a primary rain forest and agroforests in Central Amazonia[END_REF][START_REF] De Sales Dambros | Road-associated edge effects in Amazonia change termite community composition by modifying environmental conditions[END_REF][START_REF] Souza | Effects of habitat fragmentation on Amazonian termite communities[END_REF][START_REF] Davies | Environmental and spatial influences upon species composition of a termite assemblage across neotropical forest islands[END_REF]. Moreover, termites are either opportunistic or specialized prey for various invertebrates such as many species of ants [START_REF] Buczkowski | Protein marking reveals predation on termites by the woodland ant, Aphaenogaster rudis[END_REF][START_REF] Eggleton | An Introduction to Termites: Biology, Taxonomy and Functional Morphology[END_REF][START_REF] Wen | Breaking the cipher: ant eavesdropping on the variational trail pheromone of its termite prey[END_REF], spiders [START_REF] Pekár | Trophic specialisation in a predatory group: The case of prey-specialised spiders (Araneae)[END_REF] and vertebrates like anteaters, pangolins, aardvarks, etc. [START_REF] Eggleton | An Introduction to Termites: Biology, Taxonomy and Functional Morphology[END_REF]. With prey-to-predator transfer and bioaccumulation of MeHg, the health of termite predators, especially those feeding regularly on termites from heavily polluted sites may be placed at risk for MeHg contamination. As primary consumers whose body Hg concentration is the direct reflection of environmental exposure rather than prey-derived transfer, soil-feeding termites are potentially suitable candidates as bio-indicators of mercury pollution in soils of neotropical rainforest ecosystems. This study was performed in chronically contaminated sites where termites likely tolerate the pollutant. Given the potential risk of MeHg accumulation by termites on their predators, this study deserves to be extended to newly contaminated environments such as sites around artisanal and small-scale gold mining (ASGM) that are extended worldwide including in tropical areas (UN Environment, 2019;[START_REF] Gerson | Senegalese artisanal gold mining leads to elevated total mercury and methylmercury concentrations in soils, sediments, and rivers[END_REF], where termites are amongst the most representative faunal groups. If the survival of termites and their mercury-methylation capacity as shown in this study are not affected when new mercury contaminations occur, their use as bio-indicators Hg pollution could be extended to various tropical and subtropical ecosystems. Table 1. MeHg concentrations in the whole bodies of workers and nests for two soil-feeding termites, S. minutus and S. holmgreni at Crique Combat, in comparison with the nearby soils. MeHg concentrations are given for dry weights and the percentage of MeHg relative to total mercury (THg) was calculated for S. minutus nests and the nearby soils. The MeHg bioconcentration factors for the termite bodies were calculated relative to the nearby soils (BCFsoil) and the nests (BCFnest). One-way ANOVA was performed separately for each termite species and values with different bold letters differ at p <0.05.

Figures and tables

Table 2. Distribution of MeHg in termite bodies between the whole gut and the gut-free carcass of S. minutus at Petit Saut, in comparison with the concentrations in the nests and nearby soils. The concentrations of MeHg are given for dry weights and <DL indicates that the MeHg concentration in the soil was below the detection limit of the analytical instrument. Consequently, BCFs were calculated only relative to nests. Values with different bold letters differ significantly according to the one-way ANOVA at p<0.05.

Figure

  Figure 1. Concentrations of total mercury (A), mobile mercury (B) and percentage of mobile mercury (relative to total Hg, C) in nests of the soil-feeding termite

1 .

 1 Figure 1. Concentrations of total mercury (A), mobile mercury (B) and percentage of mobile mercury (relative to total Hg, C) in nests of the soil-feeding termite S. minutus at Crique Combat and the nearby soil (0-10 cm depth). The concentrations are given for dry weights. The non-parametric Kolmogorov-Smirnov test was performed separately for each mercury fraction. Different bold letters indicate significantly different values between nests and the nearby soils (p <0.05).

Figure 2 .

 2 Figure2. Linear regressions between concentrations of total mercury in the nests and in the nearby soils (A), between total mercury and mobile mercury in the nests and in the nearby soils (B), between total mercury and the percentage of mobile mercury (C), and between mobile mercury and the percentage of mobile mercury (D) in all samples. The measurements are for S. minutus at Crique Combat. Shaded areas correspond to the 95% confidence interval; R is the Spearman coefficient, p the p-value. The concentrations are given for dry weights.

Acknowledgments

The authors would like to acknowledge Virginie Roy for her help in sampling and identification of termites, Mira Toubassy for assistance in the collection of samples in the field, My-Dung Jusselme and Hervé Vincent for their contribution to the statistical analyses.

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