- Email: [email protected]
Stable isotopes reveal that the calciferous gland of earthworms is a CO2-fixing organ
ARTICLE IN PRESS
Soil Biology & Biochemistry 40 (2008) 554–557 www.elsevier.com/locate/soilbio
Short communication
Stable isotopes reveal that the calciferous gland of earthworms is a CO2-fixing organ Marı´ a Jesu´s Iglesias Brionesa,b,, Nicholas J. Ostleb, Trevor G. Piearcec a
Departamento de Ecologı´a y Biologı´a Animal, Facultad de Biologı´a, Universidad de Vigo, 36310 Vigo, Spain Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK c Department of Biological Sciences, Faculty of Science and Technology, Lancaster University, Lancaster LA1 4YQ, UK
b
Received 31 May 2007; received in revised form 17 September 2007; accepted 21 September 2007 Available online 12 October 2007
Abstract Since they were first described in 1829, earthworm calciferous glands have intrigued invertebrate anatomists and physiologists alike. These organs are present in all species of the family Lumbricidae, occurring in a range of morphological forms. A common feature of the glands is that constituent secretory cells produce a concentrated suspension of calcium carbonate. A number of possible biological roles have been suggested for the secretion (i.e. egg formation, pH buffering of the blood and ingested food, excretion and respiration) but the true function has not yet been demonstrated satisfactorily. Here, we investigated the putative respiratory function of these organs by exposing the worms to 13C-labelled CO2 and glucose and measuring tracer incorporation into the body wall, the gland tissues and the calcareous secretion. Our results support the view that these organs provide a mechanism of CO2 regulation in their tissues and that both environmental and metabolic CO2 can be fixed in this way. r 2007 Elsevier Ltd. All rights reserved. Keywords: Earthworms;
13
CO2; CO2 fixation;
13
Glucose; Stable isotopes
Earthworms (Annelida, Oligochaeta) frequently dominate soil invertebrate biomass and play a crucial role in ecosystem nutrient dynamics (e.g. Edwards and Bohlen, 1996; Ostle et al., 2007). Those species belonging to the well-represented Lumbricidae family possess a relatively complex oesophageal organ denominated calciferous gland, Kalkdru¨sen, Chylustaschen or glandes de Morren. A common feature of this structure is that its secretory cells produce a concentrated suspension of calcium carbonate (CaCO3). In certain species, this calcareous secretion passes forward to the oesophageal pouches where it precipitates as concretions of CaCO3, which are then released to the gut and finally into the soil. Several functions have been attributed to this secretion although there is no conclusive evidence in support of any proposed role. First, and most notably, the calciferous gland was considered by Darwin (1881) to be an ‘excretory organ’ that eliminates excess dietary calcium by fixing Corresponding author. Tel.: +34 986 812584; fax: +34 986 812556.
E-mail address: [email protected] (M.J.I. Briones). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.09.012
carbon dioxide (CO2) to form CaCO3. This excretion, in turn, could act to ‘neutralise’ humic acids in the ingested litter making it more nutritious to the worm (Robinet, 1883; Darwin, 1881; Harrington, 1899; Dotterweich, 1933; Piearce, 1972). In contrast, Morren (1829) and Lankester (1864) proposed the action of the gland that was related to either the reproductive (egg formation) and/or the digestion process. According to Clapare`de (1869), the mineral concretions could help to triturate the food in the gizzard whereas Michaelsen (1895, 1928) suggested that the primary function of the gland was the absorption of food products. Another interesting suggestion is that of Puytorac and Pinon (1960) and Chapron (1971) that the gland could have an important role in water regulation by acting as a barrier to reduce water loss. This idea led M’Dowall (1926) to conclude that the presence of calciferous glands in oligochaetes was an evolutionary adaptation to existence in aerobic terrestrial habitats. Others have proposed that the glands are ‘respiratory organs’, absorbing oxygen and excreting toxic CO2 accumulated in the blood or coelomic fluid by fixing it as
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
carbonate (Combault, 1909; M’Dowall, 1926; Gieschen, 1930). Voigt (1933) and Dotterweich (1933) considered that the gland was used to eliminate excess CO2 present in the soil environment, although this hypothesis was rejected by later experimental evidence which showed that only a small fraction was excreted in this way (Robertson, 1936). However, Kaestner (1967) found increased activity of the calciferous gland of Lumbricus terrestris under conditions of experimentally elevated CO2 and radiotracer studies by Ku¨hle (1980) also confirmed that some earthworm species significantly increase their gland activity with increasing environmental CO2 concentrations. In this study, we investigated the hypothesis that the calciferous gland of lumbricids is a true ‘CO2-fixing organ’ that can sequester both metabolic CO2 and CO2 present in the surrounding soil atmosphere. This was achieved by means of a short-term microcosm experiment using stable isotope 13C-labelled CO2 and glucose as tracers of atmospheric and metabolic CO2, respectively. A series of 15 plastic pots (1300 cm3) were used as experimental microcosm units with each receiving 1100 g fresh soil collected from a mown grassland on the Lancaster University Campus. Three adult earthworms (L. terrestris) were inoculated into each unit and incubated in the darkness at 15 1C for a total of 7 days. Five replicated pots were sealed with air-tight lids and received 13 C-labelled air (containing 1000 ppm, 99 at% 13CO2 at a continuous flow of 50 mL min 1) from a compressed gas cylinder (Isotech, USA) using silicone tubing, the gas leaving the circuit through an open hole made in one of the lids. The remaining units (without the lids) received an initial amount of 50 mL of labelled glucose (D-glucose–13C6; 99.9 at%) and thereafter, a daily amount of 15 mL in a concentration of 1 mg L 1 using a plastic syringe. The same volume of sterile water was added to the labelled-CO2 treatment. In addition, to counteract microbial competition for this added carbon source in five of these replicates, the soil was sterilised (autoclaved for 1 h). After 4 days, three random replicates were destructively sampled and the worms were collected. The remaining two replicates were kept in the incubators for 3 additional days, both labelled air and glucose being added only on the first of these days. On the seventh day (i.e. after 2 days without label additions), these remaining replicates were finally dismantled and the worms were removed. On each of these two sampling occasions, individual worms were dissected in deionised water to obtain samples of the body wall, the gland and the mineral concretions for 13/12C-stable isotope analyses. Nine additional worm specimens from the original grassland soil were also collected and dissected on each sampling occasion to obtain the natural abundance isotope ratios of their tissues (i.e. as an isotopic control). Tissue samples were frozen at 20 1C and freeze-dried and the concretions were air-dried prior to isotope analyses. Samples were weighed (1–2 mg), placed into tin capsules and combusted using a Eurovector elemental analyser. Resultant CO2 from combustion was analysed for delta 13C
555
using a Micromass Isoprime IRMS (Dennis Leigh, UK) at the NERC Stable Isotope Facility at CEH Lancaster. Analytical precision of o0.15 d13C (%) was obtained. We found that after 4 days of exposure to 13C-labelling treatments, the lumbricid gland and concretions incorporated most of the 13C tracer derived from the air and the glucose (Fig. 1), representing an extremely rapid and substantial CO2 binding rate. Remarkably, in the case of the body wall tissues, a small but significant amount of tracer was incorporated in those worms feeding on labelled glucose, in particular when sterile soil was used (Fig. 1a). However, the highest quantities of tracer were found in the gland tissue and, most notably, in the CaCO3 concretions (10 times higher) produced by earthworms exposed to atmospheric 13CO2 (ANOVA: Po0.0001) (Fig. 1b and c). At the same time, results from our metabolic assimilation experiment with 13C-labelled glucose showed that a substantial amount of metabolic CO2 was also converted to CaCO3. The high tracer content observed in the sterile soil treatment for all the analysed components (Fig. 1) suggests that soil microbial competition reduced the production and, therefore, binding of 13CO2 by the earthworms. After 2 days without label addition, the amount of 13C fixed in the body wall was significantly greater (ANOVA: Po0.0001) than in the control treatments (Fig. 1a). But the highest amounts of tracer were measured in the wall tissue of those worms maintained in sterile soil and fed with labelled glucose (Fig. 1a). In contrast, the calciferous gland and the mineral concretions produced by earthworms exposed to labelled air were the most enriched in 13C when compared to all of the other treatments (Figs. 1b and c), although only in the case of the gland the differences were statistically significant (Fig. 1b). Interestingly, both the calciferous glands and the concretions showed a decrease in their 13C content after 2 days without labelled glucose and reached similar values to that measured in the control worms (ANOVA: P40.05) (Fig. 1b and c) suggesting that the gland is a highly active organ and that the secretion and excretion of calcium are very rapid. The rapid incorporation of the labelled CO2 into the earthworm tissues and concretions reported here represents an extremely high CO2 binding rate. This is the first time that this direct uptake of ambient CO2 has been recorded in invertebrates and is supported by evidence that large amounts of the enzyme responsible for the elimination of the respiratory CO2 (carbonic anhydrase) are found in the gland, but are absent from the earthworm blood (Clark, 1957). The possible role of the gland in removing excess CO2 present in the blood under high CO2 concentrations (Gieschen, 1930) deserves some consideration. Authors have performed several experiments in which earthworms were exposed to high CO2 concentrations and observed an increase in the gland activity (Voigt, 1933; Dotterweich, 1933; Ku¨hle, 1980). However, the validity of conclusions drawn from these studies is questionable as it is unlikely
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
556
Body wall 10
4 days
5
c
7 days
0 d
δ13C
-5 -10 -15 -20 c
-25
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a
b
b
ab
-30 CONTROL
13GLU
13CO2
13GLU+SS
Treatment Calciferous Gland 700 4 days
600
b
7 days
500 c
δ13C
400 bc
300
ab
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ab
100 a
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Treatment Concretions 10000 9000
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5000 4000 3000
c
2000 1000
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a
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13GLU
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Treatment 13
Fig. 1. Mean7S.E. of d C values (%) of the body wall (a), calciferous gland (b) and concretion samples (c) taken after 4 days of exposure to labelled air (13CO2), labelled glucose in original soil (13Glu) and labelled glucose added to sterile soil (13Glu+SS) and after 2 days without label (end of the experiment). Different letters indicate significant differences between labelling treatments per sampling date (ANOVA; Tukey grouping, Po0.05).
that earthworms would be exposed to such high CO2 concentrations for long periods in the environment. Although, soil atmosphere is characterised by high levels of CO2, concentrations can vary with soil type, soil depth, season (Amundson and Davidson, 1990), and with microbial (Lavelle and Spain, 2001) and earthworm burrowing activities (Robertson, 1936).
On the other hand, the possibility that the CaCO3 secretion might also act, secondarily, in neutralising the acid content of the gut cannot be discarded. It has been suggested that the secretion of CaCO3 increases with ingestion of acid food (Darwin, 1881; Harrington, 1899), however, no decrease in the gland activity of worms living in alkaline soils has been observed (Bal, 1977). Therefore,
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
in these calcareous soils, the subsequent formation of mineral concretions of CaCO3 could possibly represent an excretory product, serving to eliminate excess dietary Ca (M’Dowall, 1926). A more complete picture of the true function of the gland would require further research on the relative importance of Ca excretion and CO2 excretion in a wider variety of circumstances. This production and release of CaCO3 concretions could potentially have an important role to play in soil-carbon dynamics. Individual worms can produce 1–3 concretions (4250 mm diameter) a week (Canti, 1998), many of which survive for significant time below ground, with examples commonly found in quaternary and archaeological deposits (e.g. Canti, 1998). By taking an average concretion weight of 21 mg, and assuming that 96% of this mass is CaCO3 (Robertson, 1936), it is possible to estimate that an earthworm population of 100 individuals m 2 would fix 12.5 g C m 2 y 1. Although densities of 100–500 earthworms m 2 have been recorded (Lavelle and Spain, 2001) in neutral and high-pH soils, an equivalent of 10–20 large L. terrestris per square metre is often recorded. This average population density would yield 12.5–25 kg C ha 1 y 1, suggesting that this mechanism of C sequestration could be extremely important. Acknowledgements We thank J. Poskitt and S. Oakley for laboratory assistance and Dr. L. Gago-Duport for his valuable comments. References Amundson, R.G., Davidson, E.A., 1990. Carbon dioxide and nitrogenous gases in the soil atmosphere. Journal of Geochemical Exploration 38, 13–41. Bal, L., 1977. The formation of carbonate nodules and intercalary crystals in the soil by the earthworm Lumbricus rubellus. Pedobiologia 17, 102–106. Canti, M., 1998. Origin of calcium carbonate granules found in buried soils and quaternary deposits. Boreas 27, 275–288. Chapron, C., 1971. E´tude du role de la glande de Morren des Lombriciens dans la re´gulation de l’eau. Journal de Miscroscopie 10, 351–356. Clapare`de, E., 1869. Histologische Untersuchungen u¨ber den Regenwurm. Zeitschrift fu¨r Wissenschaftliche Zoologie 19, 563–624. Clark, A.M., 1957. The distribution of carbonic anhydrase in the earthworm and snail. Australian Journal of Science 19, 205–207.
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Combault, A., 1909. Contribution a` l’e´tude de la respiration et de la circulation des Lombriciens. Journal of Anatomy 45, 358–399, 474–534. Darwin, C., 1881. The Formation of Vegetable Mould, Through The Action of Worms, with Observations on Their Habits. John Murray, London, 153pp. Dotterweich, H., 1933. Die Funktion tierischer Kalkablagerungen als Pufferreserve im Dienste der Reaktionsregulation. Die Kalkdru¨sen des Regenwurms. Pflu¨gers Archiv fu¨r geschicte der Physiologie 232, 263–286. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms. Chapman & Hall, London, 426pp. Gieschen, A., 1930. Die physiologische Bedeutung der Kalkdru¨sen. Zoologische Jahrbu¨cher-Abteilung fu¨r Allgemeine Zoologie und Physiologie der Tiere 52, 677–708. Harrington, N.R., 1899. The calciferous glands of the earthworm, with appendix on the circulation. Journal of Morphology 15 (Suppl.), 105–168. Kaestner, A., 1967. Invertebrate Zoology. Interscience Publishers, New York, 537pp. Ku¨hle, J.C., 1980. Vergleichende Untersuchungen zur Funktion der Kalkdru¨se verschiedener Regenwurmarten bei unterschiedlicher CO2Atmospha¨re. Verhandlungen der Gesellschaft fu¨r O¨kologie 8, 411–415. Lankester, E.R., 1864. The anatomy of the earthworm. Quarterly Journal of Microscopical Science 4, 258–268. Lavelle, P., Spain, A.V., 2001. Soil Ecology. Kluwer Academic Publishers, Dordrecht, 654pp. M’Dowall, J., 1926. Preliminary work towards a morphological and physiological study of the calciferous glands of the earthworm. Proceedings of the Royal Physical Society of Edinburgh 21, 65–72. Michaelsen, W., 1895. Zur Kenntnis der Oligochaeten. Abhandlungen und Verhandlungen des Naturwissenschaftlichen Vereins, Hamburg 13, 1–37. Michaelsen, W., 1928. Oligocheata. In: Ku¨kenthal, W., Krumbach, T. (Eds.), Handbuch der Zoologie. de Gruyter, Berlin, pp. 1–118. Morren, C.F.A., 1829. De Lumbrici terrestris Historia Naturali Necnon Anatomia Tractatus, Bruxelles, 280pp. Ostle, N., Briones, M.J.I., Ineson, P., Cole, L., Staddon, P., Sleep, D., 2007. Isotopic detection of recent photosynthate carbon flow into grassland rhizosphere fauna. Soil Biology & Biochemistry 39, 768–777. Piearce, T.G., 1972. The calcium relations of selected Lumbricidae. Journal of Animal Ecology 41, 167–188. Puytorac, P., Pinon, M., 1960. E´tude des variations du poids de Lumbricus terrestris (ver Oligoche`te) immerge`s dans l’eau. Archives de Zoologie Experimentale et Generale 99, 23–43. Robertson, J.D., 1936. The function of the calciferous glands of earthworms. Journal of Experimental Biology 13, 279–297. Robinet, C., 1883. Recherches physiologique sur la secretion des glandes de Morren du Lumbricus terrestris. Comptes Rendus des Sceances de l’Academie des Sciences de Paris 97, 192–194. Voigt, O., 1933. Die Funktion der Regenwurm-Kalkdru¨sen. Zoologische Jahrbu¨cher-Abteilung fur Allgemeine Zoologie und Physiologie der Tiere 52, 677–708.
Soil Biology & Biochemistry 40 (2008) 554–557 www.elsevier.com/locate/soilbio
Short communication
Stable isotopes reveal that the calciferous gland of earthworms is a CO2-fixing organ Marı´ a Jesu´s Iglesias Brionesa,b,, Nicholas J. Ostleb, Trevor G. Piearcec a
Departamento de Ecologı´a y Biologı´a Animal, Facultad de Biologı´a, Universidad de Vigo, 36310 Vigo, Spain Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK c Department of Biological Sciences, Faculty of Science and Technology, Lancaster University, Lancaster LA1 4YQ, UK
b
Received 31 May 2007; received in revised form 17 September 2007; accepted 21 September 2007 Available online 12 October 2007
Abstract Since they were first described in 1829, earthworm calciferous glands have intrigued invertebrate anatomists and physiologists alike. These organs are present in all species of the family Lumbricidae, occurring in a range of morphological forms. A common feature of the glands is that constituent secretory cells produce a concentrated suspension of calcium carbonate. A number of possible biological roles have been suggested for the secretion (i.e. egg formation, pH buffering of the blood and ingested food, excretion and respiration) but the true function has not yet been demonstrated satisfactorily. Here, we investigated the putative respiratory function of these organs by exposing the worms to 13C-labelled CO2 and glucose and measuring tracer incorporation into the body wall, the gland tissues and the calcareous secretion. Our results support the view that these organs provide a mechanism of CO2 regulation in their tissues and that both environmental and metabolic CO2 can be fixed in this way. r 2007 Elsevier Ltd. All rights reserved. Keywords: Earthworms;
13
CO2; CO2 fixation;
13
Glucose; Stable isotopes
Earthworms (Annelida, Oligochaeta) frequently dominate soil invertebrate biomass and play a crucial role in ecosystem nutrient dynamics (e.g. Edwards and Bohlen, 1996; Ostle et al., 2007). Those species belonging to the well-represented Lumbricidae family possess a relatively complex oesophageal organ denominated calciferous gland, Kalkdru¨sen, Chylustaschen or glandes de Morren. A common feature of this structure is that its secretory cells produce a concentrated suspension of calcium carbonate (CaCO3). In certain species, this calcareous secretion passes forward to the oesophageal pouches where it precipitates as concretions of CaCO3, which are then released to the gut and finally into the soil. Several functions have been attributed to this secretion although there is no conclusive evidence in support of any proposed role. First, and most notably, the calciferous gland was considered by Darwin (1881) to be an ‘excretory organ’ that eliminates excess dietary calcium by fixing Corresponding author. Tel.: +34 986 812584; fax: +34 986 812556.
E-mail address: [email protected] (M.J.I. Briones). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.09.012
carbon dioxide (CO2) to form CaCO3. This excretion, in turn, could act to ‘neutralise’ humic acids in the ingested litter making it more nutritious to the worm (Robinet, 1883; Darwin, 1881; Harrington, 1899; Dotterweich, 1933; Piearce, 1972). In contrast, Morren (1829) and Lankester (1864) proposed the action of the gland that was related to either the reproductive (egg formation) and/or the digestion process. According to Clapare`de (1869), the mineral concretions could help to triturate the food in the gizzard whereas Michaelsen (1895, 1928) suggested that the primary function of the gland was the absorption of food products. Another interesting suggestion is that of Puytorac and Pinon (1960) and Chapron (1971) that the gland could have an important role in water regulation by acting as a barrier to reduce water loss. This idea led M’Dowall (1926) to conclude that the presence of calciferous glands in oligochaetes was an evolutionary adaptation to existence in aerobic terrestrial habitats. Others have proposed that the glands are ‘respiratory organs’, absorbing oxygen and excreting toxic CO2 accumulated in the blood or coelomic fluid by fixing it as
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
carbonate (Combault, 1909; M’Dowall, 1926; Gieschen, 1930). Voigt (1933) and Dotterweich (1933) considered that the gland was used to eliminate excess CO2 present in the soil environment, although this hypothesis was rejected by later experimental evidence which showed that only a small fraction was excreted in this way (Robertson, 1936). However, Kaestner (1967) found increased activity of the calciferous gland of Lumbricus terrestris under conditions of experimentally elevated CO2 and radiotracer studies by Ku¨hle (1980) also confirmed that some earthworm species significantly increase their gland activity with increasing environmental CO2 concentrations. In this study, we investigated the hypothesis that the calciferous gland of lumbricids is a true ‘CO2-fixing organ’ that can sequester both metabolic CO2 and CO2 present in the surrounding soil atmosphere. This was achieved by means of a short-term microcosm experiment using stable isotope 13C-labelled CO2 and glucose as tracers of atmospheric and metabolic CO2, respectively. A series of 15 plastic pots (1300 cm3) were used as experimental microcosm units with each receiving 1100 g fresh soil collected from a mown grassland on the Lancaster University Campus. Three adult earthworms (L. terrestris) were inoculated into each unit and incubated in the darkness at 15 1C for a total of 7 days. Five replicated pots were sealed with air-tight lids and received 13 C-labelled air (containing 1000 ppm, 99 at% 13CO2 at a continuous flow of 50 mL min 1) from a compressed gas cylinder (Isotech, USA) using silicone tubing, the gas leaving the circuit through an open hole made in one of the lids. The remaining units (without the lids) received an initial amount of 50 mL of labelled glucose (D-glucose–13C6; 99.9 at%) and thereafter, a daily amount of 15 mL in a concentration of 1 mg L 1 using a plastic syringe. The same volume of sterile water was added to the labelled-CO2 treatment. In addition, to counteract microbial competition for this added carbon source in five of these replicates, the soil was sterilised (autoclaved for 1 h). After 4 days, three random replicates were destructively sampled and the worms were collected. The remaining two replicates were kept in the incubators for 3 additional days, both labelled air and glucose being added only on the first of these days. On the seventh day (i.e. after 2 days without label additions), these remaining replicates were finally dismantled and the worms were removed. On each of these two sampling occasions, individual worms were dissected in deionised water to obtain samples of the body wall, the gland and the mineral concretions for 13/12C-stable isotope analyses. Nine additional worm specimens from the original grassland soil were also collected and dissected on each sampling occasion to obtain the natural abundance isotope ratios of their tissues (i.e. as an isotopic control). Tissue samples were frozen at 20 1C and freeze-dried and the concretions were air-dried prior to isotope analyses. Samples were weighed (1–2 mg), placed into tin capsules and combusted using a Eurovector elemental analyser. Resultant CO2 from combustion was analysed for delta 13C
555
using a Micromass Isoprime IRMS (Dennis Leigh, UK) at the NERC Stable Isotope Facility at CEH Lancaster. Analytical precision of o0.15 d13C (%) was obtained. We found that after 4 days of exposure to 13C-labelling treatments, the lumbricid gland and concretions incorporated most of the 13C tracer derived from the air and the glucose (Fig. 1), representing an extremely rapid and substantial CO2 binding rate. Remarkably, in the case of the body wall tissues, a small but significant amount of tracer was incorporated in those worms feeding on labelled glucose, in particular when sterile soil was used (Fig. 1a). However, the highest quantities of tracer were found in the gland tissue and, most notably, in the CaCO3 concretions (10 times higher) produced by earthworms exposed to atmospheric 13CO2 (ANOVA: Po0.0001) (Fig. 1b and c). At the same time, results from our metabolic assimilation experiment with 13C-labelled glucose showed that a substantial amount of metabolic CO2 was also converted to CaCO3. The high tracer content observed in the sterile soil treatment for all the analysed components (Fig. 1) suggests that soil microbial competition reduced the production and, therefore, binding of 13CO2 by the earthworms. After 2 days without label addition, the amount of 13C fixed in the body wall was significantly greater (ANOVA: Po0.0001) than in the control treatments (Fig. 1a). But the highest amounts of tracer were measured in the wall tissue of those worms maintained in sterile soil and fed with labelled glucose (Fig. 1a). In contrast, the calciferous gland and the mineral concretions produced by earthworms exposed to labelled air were the most enriched in 13C when compared to all of the other treatments (Figs. 1b and c), although only in the case of the gland the differences were statistically significant (Fig. 1b). Interestingly, both the calciferous glands and the concretions showed a decrease in their 13C content after 2 days without labelled glucose and reached similar values to that measured in the control worms (ANOVA: P40.05) (Fig. 1b and c) suggesting that the gland is a highly active organ and that the secretion and excretion of calcium are very rapid. The rapid incorporation of the labelled CO2 into the earthworm tissues and concretions reported here represents an extremely high CO2 binding rate. This is the first time that this direct uptake of ambient CO2 has been recorded in invertebrates and is supported by evidence that large amounts of the enzyme responsible for the elimination of the respiratory CO2 (carbonic anhydrase) are found in the gland, but are absent from the earthworm blood (Clark, 1957). The possible role of the gland in removing excess CO2 present in the blood under high CO2 concentrations (Gieschen, 1930) deserves some consideration. Authors have performed several experiments in which earthworms were exposed to high CO2 concentrations and observed an increase in the gland activity (Voigt, 1933; Dotterweich, 1933; Ku¨hle, 1980). However, the validity of conclusions drawn from these studies is questionable as it is unlikely
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
556
Body wall 10
4 days
5
c
7 days
0 d
δ13C
-5 -10 -15 -20 c
-25
a
a
b
b
ab
-30 CONTROL
13GLU
13CO2
13GLU+SS
Treatment Calciferous Gland 700 4 days
600
b
7 days
500 c
δ13C
400 bc
300
ab
200
b
ab
100 a
a
0 -100
CONTROL
13GLU
13CO2
13GLU+SS
Treatment Concretions 10000 9000
4 days
8000
7 days
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a b
δ13C
6000
d
5000 4000 3000
c
2000 1000
a a
a
a
0 -1000
CONTROL
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13GLU
13GLU+SS
Treatment 13
Fig. 1. Mean7S.E. of d C values (%) of the body wall (a), calciferous gland (b) and concretion samples (c) taken after 4 days of exposure to labelled air (13CO2), labelled glucose in original soil (13Glu) and labelled glucose added to sterile soil (13Glu+SS) and after 2 days without label (end of the experiment). Different letters indicate significant differences between labelling treatments per sampling date (ANOVA; Tukey grouping, Po0.05).
that earthworms would be exposed to such high CO2 concentrations for long periods in the environment. Although, soil atmosphere is characterised by high levels of CO2, concentrations can vary with soil type, soil depth, season (Amundson and Davidson, 1990), and with microbial (Lavelle and Spain, 2001) and earthworm burrowing activities (Robertson, 1936).
On the other hand, the possibility that the CaCO3 secretion might also act, secondarily, in neutralising the acid content of the gut cannot be discarded. It has been suggested that the secretion of CaCO3 increases with ingestion of acid food (Darwin, 1881; Harrington, 1899), however, no decrease in the gland activity of worms living in alkaline soils has been observed (Bal, 1977). Therefore,
ARTICLE IN PRESS M.J.I. Briones et al. / Soil Biology & Biochemistry 40 (2008) 554–557
in these calcareous soils, the subsequent formation of mineral concretions of CaCO3 could possibly represent an excretory product, serving to eliminate excess dietary Ca (M’Dowall, 1926). A more complete picture of the true function of the gland would require further research on the relative importance of Ca excretion and CO2 excretion in a wider variety of circumstances. This production and release of CaCO3 concretions could potentially have an important role to play in soil-carbon dynamics. Individual worms can produce 1–3 concretions (4250 mm diameter) a week (Canti, 1998), many of which survive for significant time below ground, with examples commonly found in quaternary and archaeological deposits (e.g. Canti, 1998). By taking an average concretion weight of 21 mg, and assuming that 96% of this mass is CaCO3 (Robertson, 1936), it is possible to estimate that an earthworm population of 100 individuals m 2 would fix 12.5 g C m 2 y 1. Although densities of 100–500 earthworms m 2 have been recorded (Lavelle and Spain, 2001) in neutral and high-pH soils, an equivalent of 10–20 large L. terrestris per square metre is often recorded. This average population density would yield 12.5–25 kg C ha 1 y 1, suggesting that this mechanism of C sequestration could be extremely important. Acknowledgements We thank J. Poskitt and S. Oakley for laboratory assistance and Dr. L. Gago-Duport for his valuable comments. References Amundson, R.G., Davidson, E.A., 1990. Carbon dioxide and nitrogenous gases in the soil atmosphere. Journal of Geochemical Exploration 38, 13–41. Bal, L., 1977. The formation of carbonate nodules and intercalary crystals in the soil by the earthworm Lumbricus rubellus. Pedobiologia 17, 102–106. Canti, M., 1998. Origin of calcium carbonate granules found in buried soils and quaternary deposits. Boreas 27, 275–288. Chapron, C., 1971. E´tude du role de la glande de Morren des Lombriciens dans la re´gulation de l’eau. Journal de Miscroscopie 10, 351–356. Clapare`de, E., 1869. Histologische Untersuchungen u¨ber den Regenwurm. Zeitschrift fu¨r Wissenschaftliche Zoologie 19, 563–624. Clark, A.M., 1957. The distribution of carbonic anhydrase in the earthworm and snail. Australian Journal of Science 19, 205–207.
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