Abstract
This study demonstrates that 31P and 1H high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR), a recently developed NMR technique, can be applied to the in vivo analysis of metabolites from an unstarved earthworm. The endogeic earthworm Aporrectodea caliginosa was cut into lengths and then the different body parts (anterior, middle, posterior) with the gut content were analyzed. With the HR-MAS NMR, metabolites show well-resolved signals, whereas, with conventional NMR, spectra are highly dependent on the gut content. 31P HR-MAS NMR has been used to evaluate the effect of an acute exposure of the earthworm to glyphosate. Our observations support a low toxicity of the herbicide and suggest that glyphosate could be trapped in the cutaneous mucus. Earthworms could therefore play a role in horizontal dispersion and stabilization of glyphosate in the drilosphere. Phosphorylated metabolites, such as phospholombricine and lombricine, were clearly identified and their amount measured during experiments. The 1H HR-MAS NMR method offers the opportunity to measure, on the same sample and simultaneously, both the hydrosoluble metabolites and lipids. The data on lipid location and relative succinate concentration shed light on the physiological and metabolic functions of the different body parts of the earthworm.
Similar content being viewed by others
References
Bundy JG, Spurgeon DJ, Svendsen C, Hankard PK, Osborn D, Lindon JC, Nicholson JK (2002) Earthworm species of the genus Eisenia can be phenotypically differentiated by metabolic profiling. FEBS Lett 521:115–120
Bundy JG, Spurgeon DJ, Svendsen C, Hankard PK, Weeks JM, Osborn D, Lindon JC, Nicholson JK (2004) Environmental metabolomics: applying combination biomarker analysis in earthworms at a metal contaminated site. Ecotoxicology 13:797–806
Dalby PR, Baker GH, Smith SE (1995) Glyphosate, 2-4-DB and dimethoate: effects on earthworm survival and growth. Soil Biol Biochem 27:1661–1662
Desmoulin F, Cozzone PJ, Canioni P (1987) Phosphorus-31 nuclear magnetic resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver. Eur J Biochem 162:151–159
Edwards CA (1983) Earthworm ecology in cultivated soils. In: Stachell JE (ed) Earthworm ecology from Darwin to vermiculture. Chapman & Hall, London, pp 123–137
Ennor AH, Rosenberg H, Rossiter RJ, Beatty IM, Gaffney T (1960) The isolation and characterization of D-serine ethanolamine phosphodiester from earthworms. Biochem J 75:179–182
Fan TWM (1996) Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Prog Nucl Magn Reson Spectrosc 28:161–219
Farenhorst A, Topp E, Bowman BT, Tomlin AD (2000) Earthworm burrowing and feeding activity and the potential for atrazine transport by preferential flow. Soil Biol Biochem 32:479–488
Gibb JOT, Holmes E, Nicholson JK, Weeks JM (1997a) Proton NMR spectroscopic studies on tissue extracts of invertebrate species with pollution indicator potential. Comp Biochem Physiol 118B:587–598
Gibb JOT, Svendsen C, Weeks JM, Nicholson JK (1997b) 1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu(II) exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms. Biomarkers 2:295–302
Horn MA, Schramm A, Drake HL (2003) The earthworm gut: an ideal habitat for ingested N2O-producing microorganisms. Appl Environ Microbiol 69:1662–1669
Ihssen J, Horn MA, Matthies C, Göβner A, Schramm A, Drake HL (2003) N2O-Producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria. Appl Environ Microbiol 69:1655–1661
Jégou D, Schrader S, Diestel H, Cluzeau D (2001) Morphological, physical and biochemical characteristics of burrow walls formed by earthworms. Appl Soil Ecol 17:165–174
Kudzin ZH, Gralak DK, Drabowicz J, Luczak J (2002) Novel approach for the simultaneous analysis of glyphosate and its metabolites. J Chromatogr A 947:129–141
Lanno R, Wells J, Conder J, Bradham K, Basta N (2004) The bioavailability of chemicals in soil for earthworms. Ecotoxicol Environ Saf 57:39–47
Lindon JC, Nicholson JK, Holmes E, Everett JR (2000) Metabolomics: metabolic processes studied by NMR spectroscopy of biofluid. Concepts Magn Reson 12(5):289–320
Moka D, Vorrheuter R, Schicha H, Spraul M, Humpfer E, Lipinski M, Foxall PJD, Nicholson JK, Lindon JC (1997) Magic angle spinning proton nuclear magnetic resonance spectroscopic analysis of intact kidney tissue samples. Anal Commun 34:107–109
Norsworthy JK, Frederick JR (2005) Integrated weed management strategies for maize (Zea mays) production on the south-eastern coastal plains of North America. Crop Prot 24:119–126
Savigny JC (1826) In: Cuvier, « Analyse des travaux de l’Académie Royale des Sciences pendant l’année 1821, partie physique » Mem Acad Roy Sci Inst Fr 5:176–184
Simpson AJ, Kincery WL, Shaw DR, Spraul M, Humpfer E, Dvortsak P (2001) The application of 1H HR-MAS spectroscopy for the study of structures and associations of organic components at the solid-aqueous interface of a whole soil. Environ Sci Technol 35:3321–3325
Stehouwer RC, Dick WA, Traina SJ (1994) Sorption and retention of herbicides in vertically oriented earthworm and artificial burrows. J Environ Qual 23:286–292
Tyagi RK, Azrad A, Degani H, Salomon Y (1996) Simultaneous extraction of cellular lipids and water soluble metabolites: evaluation by NMR spectroscopy. Magn Reson Med 35:194–200
Van Den Thillart G, Van Waarde A (1996) Nuclear magnetic resonance spectroscopy of living systems: applications in comparative physiology. Physiol Rev 76:799–837
Van Waarde A, Van Den Thillart G, Verhagen M, Erkelens C, Addink A, Lugtenburg J (1990) Direct observation of the phosphate acceptor and phosphagen pool sizes in vivo. Am J Physiol 258:R1132–R1139
Warne MA, Lenz EM, Osborn D, Weeks JM, Nicholson JK (2001) Comparative biochemistry and short-term starvation effects on the earthworms Eisenia veneta and Lumbricus terrestris studied by 1H NMR spectroscopy and pattern recognition. Soil Biol Biochem 33:1171–1180
Yu Y, Zhou QX (2004) Adsorption characteristics of pesticides methamidophos and glyphosate by two soils. Chemosphere 58:811–816
Acknowledgements
We thank Dr. Marcus A. Horn (University of Bayreuth, Germany), for the fruitful discussion on earthworm gut microbiota, and Laura Desmoulin, for help to collect earthworms.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bon, D., Gilard, V., Massou, S. et al. In vivo 31P and 1H HR-MAS NMR spectroscopy analysis of the unstarved Aporrectodea caliginosa (Lumbricidae). Biol Fertil Soils 43, 191–198 (2006). https://doi.org/10.1007/s00374-006-0092-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00374-006-0092-7