Skip to main content

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

Abstract

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439–455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8–10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. An JH, Blackwell TK (2003) SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev 17:1882–1893

    Article  CAS  Google Scholar 

  2. Anbalagan C, Lafayette I, Antoniou-Kourounioti M, Haque M, King J, Johnsen B, Baillie D, Gutierrez C, Rodriguez Martin J, de Pomerai D (2012) Transgenic nematodes as biosensors for metal stress in soil pore water samples. Ecotoxicology 21:439–455

    Article  CAS  Google Scholar 

  3. Bargmann CI (2006) Chemosensation in C. elegans. In: The C. elegans Research Community (ed) Wormbook. doi:10.1895/wormbook.1.123.1. http://www.wormbook.org. Accessed 10 July 2012

  4. Boyd WA, Smith MV, Kissling GE, Rice JR, Snyder DW, Portier CJ, Freedman JH (2009) Application of a mathematical model to describe the effects of chlorpyrifos on Caenorhabditis elegans development. PLoS One 4:e7042

    Article  Google Scholar 

  5. Boyd WA, Smith MV, Kissling GE, Freedman JH (2010) Medium- and high-throughput screening of neurotoxicants using C. elegans. Neurotoxicol Teratol 32:68–73

    Article  CAS  Google Scholar 

  6. Candido EPM, Jones D (1996) Transgenic Caenorhabditis elegans strains as biosensors. Trends Biotechnol 14:125–129

    Article  CAS  Google Scholar 

  7. Casida JE (2009) Pest toxicology: the primary mechanisms of pesticide action. Chem Res Toxicol 22:609–619

    Article  CAS  Google Scholar 

  8. C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018

    Article  Google Scholar 

  9. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805

    Article  CAS  Google Scholar 

  10. Chu K, Chow K (2002) Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative. Aquat Toxicol 61:53–64

    Article  Google Scholar 

  11. Cioci LK, Qiu L, Freedman JH (2000) Transgenic strains of the nematode Caenorhabditis elegans as biomonitors of metal contamination. Environ Toxicol Chem 19:2122–2129

    CAS  Google Scholar 

  12. David HE, Dawe AS, de Pomerai DI, Jones D, Candido EPM, Daniells C (2003) Construction and evaluation of a transgenic hsp16-GFP-lacZ Caenorhabditis elegans strain for environmental monitoring. Environ Toxicol Chem 22:111–118

    CAS  Google Scholar 

  13. de Pomerai DI (1996) Heat shock proteins as biomarkers of pollution. Hum Exp Toxicol 15:279–285

    Article  Google Scholar 

  14. Derry WB, Putzke AP, Rothman JH (2001) Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science 294:591–595

    Article  CAS  Google Scholar 

  15. Dhawan R, Dusenbery D, Williams P (1999) Comparison of lethality, reproduction and behaviour as toxicological endpoints in the nematode Caenorhabditis elegans. J Toxicol Environ Health A 58:451–462

    Article  CAS  Google Scholar 

  16. Freedman JH, Slice LW, Dixon D, Fire A, Rubin CS (1993) The novel metallothionein genes of Caenorhabditis elegans. J Biol Chem 268:2554–2564

    CAS  Google Scholar 

  17. Gomez-Eyles JL, Svendsen C, Lister L, Martin H, Hodson ME, Spurgeon DJ (2008) Measuring and modelling mixture toxicity of imidacloprid and thiacloprid on Caenorhabditis elegans and Eisenia foetida. Ecotoxicol Environ Saf 72:71–79

    Article  Google Scholar 

  18. Guven K, Duce J, de Pomerai DI (1994) Evaluation of a stress-inducible transgenic nematode strain for rapid aquatic toxicity testing. Aquat Toxicol 29:119–137

    Article  CAS  Google Scholar 

  19. Guven K, Duce J, de Pomerai DI (1995) Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans. Comp Biochem Physiol 110C:61–70

    CAS  Google Scholar 

  20. Hasegawa K, Miwa S, Isomura K, Tsutsumiuchi K, Taniguchi H, Miwa J (2008) Acrylamide-responsive genes in the nematode Caenorhabditis elegans. Toxicol Sci 101:215–225

    Article  CAS  Google Scholar 

  21. Haynes CM, Ron D (2010) The mitochondrial UPR—protecting organelle protein homeostasis. J Cell Sci 123:3849–3855

    Article  CAS  Google Scholar 

  22. Hunt-Newbury R, Viveiros R, Johnsen R, Mah A, Anastis D, Fang L, Halfnight E, Lee D, Lin J, Lorch A, McKay S, Okada HM, Pan J, Schultz AK, Tu D, Wong K, Zhao Z, Alexeyenko A, Burglin T, Sonnhammer E, Schnabel R, Jones SJ, Marra MA, Baillie DL, Moerman DG (2007) High throughput in vivo analysis of gene expression in Caenorhabditis elegans. PLoS Biol 5:e237

    Article  Google Scholar 

  23. Jadhav KB, Rajini PS (2009) Neurophysiological alterations in Caenorhabditis elegansexposed to dichlorvos, an organophosphorus insecticide. Pestic Biochem Physiol 94:79–85

    Article  CAS  Google Scholar 

  24. Jones D, Candido EPM (1999) Feeding is inhibited by sublethal concentrations of toxicants and by heat stress in the nematode Caenorhabditis elegans: relationship to the cellular stress response. J Exp Zool 284:147–157

    Article  CAS  Google Scholar 

  25. Kamath RS, Fraser A, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231–237

    Article  CAS  Google Scholar 

  26. Kenyon CJ (2010) The genetics of ageing. Nature 464:504–512

    Article  CAS  Google Scholar 

  27. Leung MCK, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, Meyer JN (2008) Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology. Toxicol Sci 106:5–28

    Article  CAS  Google Scholar 

  28. Lindblom TH, Pierce GJ, Sluder AE (2001) A C. elegans orphan nuclear receptor contributes to xenobiotic resistance. Curr Biol 11:864–868

    Article  CAS  Google Scholar 

  29. Link C, Cypser J, Johnson C, Johnson T (1999) Direct observation of stress response in Caenorhabditis elegans using a reporter trans-gene. Cell Stress Chaperones 4:235–242

    Article  CAS  Google Scholar 

  30. Lopez Arias M, Rodriguez JA (2005) Metales pesados, materias organica y otros parametros de la capa superficial de los suelos agricolas y de pastos de la Espana peninsular. I: Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria y Ministerio de Educacion y Ciencia (ed) Resultados globales. Ministerio de Educacion y Ciencia, Madrid, p 249

  31. Ma H, Glenn TC, Jagoe CH, Jones KL, Williams PL (2009) A transgenic strain of the nematode Caenorhabditis elegans as a biomonitor for heavy metal contamination. Environ Toxicol Chem 28:1311–1318

    Article  CAS  Google Scholar 

  32. Melstrom P, Williams PL (2007) Reversible AChE inhibitors in C. elegans vs. rats, mice. Biochem Biophys Res Commun 357:200–205

    Article  CAS  Google Scholar 

  33. Menzel R, Bogaert T, Achazi R (2001) A systematic gene expression screen of Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic inducible. Arch Biochem Biophys 395:158–168

    Article  CAS  Google Scholar 

  34. Menzel R, Yeo HL, Rienau S, Li S, Steinberg CEW, Sturzenbaum SR (2007) Cytochrome P450 s and short-chain dehydrogenases mediate the toxicogenomic response of PCB52 in the nematode Caenorhabditis elegans. J Mol Biol 370:1–13

    Article  CAS  Google Scholar 

  35. Moilanen LH, Fukushige T, Freedman JH (1999) Regulation of metallothionein gene transcription: identification of upstream regulatory element and transcription factors responsible for cell-specific expression of metallothionein genes from Caenorhabditis elegans. J Biol Chem 274:29655–29665

    Article  CAS  Google Scholar 

  36. Mutwakil MHAZ, Reader JP, Holdich DM, Smithurst PR, Candido EPM, Jones D, de Pomerai DI (1997) Use of stress-inducible transgenic nematodes as biomarkers of heavy metal pollution in water samples from an English river system. Arch Environ Contam Toxicol 32:146–153

    Article  CAS  Google Scholar 

  37. Negga R, Rudd DA, Davis NS, Justice AN, Hatfield HE, Valente AL, Fields AS, Fitsanakis VA (2011) Exposure to Mn/Zn ethylene-bis-dithiocarbamate and glyphosate pesticides leads to neurodegeneration in Caenorhabditis elegans. NeuroToxicology 32:331–341

    Article  CAS  Google Scholar 

  38. Power RS, de Pomerai DI (1999) Effect of single and paired metal inputs in soil on a stress-inducible transgenic nematode. Arch Environ Contam Toxicol 37:503–511

    Article  CAS  Google Scholar 

  39. Power RS, de Pomerai DI (2001) Application of a stress-inducible nematode to soil biomonitoring. In: Rainbow PS, Hopkin SP, Crane M (eds) Forecasting the environmental fate and effects of chemicals. Wiley, Chichester, pp 125–138

  40. Rajini PS, Melstrom P, Williams P (2008) A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides. J Toxicol Environ Health A 71:1043–1050

    Article  CAS  Google Scholar 

  41. Rodríguez JA, Lopez Arias M, Grau Corbi JM (2006) Heavy metals contents in agricultural topsoils in the Ebro basin (Spain). Application of the multivariate geo-statistical methods to study spatial variations. Environ Pollut 144:1001–1012

    Article  Google Scholar 

  42. Roh J-Y, Choi J (2008) Ecotoxicological evaluation of chlorpyrifos exposure on the nematode Caenorhabditis elegans. Ecotoxicol Environ Saf 71:483–489

    Article  CAS  Google Scholar 

  43. Roh J-Y, Choi J (2011) Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans. Chemosphere 84:1356–1361

    Article  CAS  Google Scholar 

  44. Shashikumar S, Rajini PS (2010) Cypermethrin elicited responses in heat shock protein and feeding in Caenorhabditis elegans. Ecotoxicol Environ Saf 73:1057–1062

    Article  CAS  Google Scholar 

  45. Sochova I, Hofman J, Holoubek I (2007) Effects of seven organic pollutants on soil nematode Caenorhabditis elegans. Environ Int 33:798–804

    Article  CAS  Google Scholar 

  46. Stringham E, Candido EPM (1994) Transgenic hsp16-lacZ strains of the soil nematode Caenorhabditis elegans as biological monitors of environmental stress. Environ Toxicol Chem 13:1211–1220

    CAS  Google Scholar 

  47. Sulston JE, Schierenberg E, White JG, Thomson JN (1983) The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 100:64–119

    Article  CAS  Google Scholar 

  48. Swain SC, Keusekotten K, Baumeister R, Sturzenbaum SR (2004) C. elegans metallothioneins: new insights into the phenotypic effects of cadmium toxicosis. J Mol Biol 341:951–959

    Article  CAS  Google Scholar 

  49. Thompson G, de Pomerai DI (2005) Toxicity of short-chain alcohols to the nematode Caenorhabditis elegans: a comparison of endpoints. J Biochem Mol Toxicol 19:87–95

    Article  CAS  Google Scholar 

  50. Traunspurger W, Haitzer M, Hoss S, Beier S, Ahle W, Steinberg C (1997) Ecotoxicological assessment of aquatic sediments with Caenorhabditis elegans (Nematoda)—a method for testing liquid medium and whole-sediment samples. Environ Toxicol Chem 16:245–250

    CAS  Google Scholar 

  51. USEPA (1996) EPA standard method 3540C: soxhlet extraction. US Environmental Protection Agency, Washington DC

    Google Scholar 

  52. Vinogradova I, Cook A, Holden-Dye L (2006) The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans. Invert Neurosci 6:57–68

    Article  CAS  Google Scholar 

  53. Vinuela A, Snoek LB, Riksen JAG, Kammenga JE (2010) Genome-wide expression analysis in response to organophosphorus pesticide chlorpyrifos and diazinon in C. elegans. PLoS One 5:e12145

    Article  Google Scholar 

  54. Williams PL, Dusenbery DB (1990) Aquatic toxicity testing using the nematode Caenorhabditis elegans. Environ Toxicol Chem 9:1285–1290

    CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr Bob Johnsen and Professor David Baillie for providing the BC transgenic strains from the Baillie Genome GFP project (Simon Fraser University, Burnaby, Vancouver, Canada), Professors Chris Link, Cynthia Kenyon, Joel Rothman and Ralph Menzel for various GFP transgenic strains (see “Materials and methods” section), Dr Liz Bailey (Environmental Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington, UK) for carrying out the metal determinations, as well as Luis Cuadra and Elena Fernandez (Department of Environmental Contamination, Instituto de Ciencias Agrarias (ICA)-CSIC, Madrid, Spain) for preparing the organic extracts. This study was funded by the British Council through Major Award number MA-05 to DdeP under its UK-India Education and Research Initiative (UK-IERI).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Affiliations

Authors

Corresponding author

Correspondence to David I. De Pomerai.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (XLSX 65 kb)

Supplementary material 2 (TIFF 3138 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Anbalagan, C., Lafayette, I., Antoniou-Kourounioti, M. et al. Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils. Ecotoxicology 22, 72–85 (2013). https://doi.org/10.1007/s10646-012-1004-2

Download citation

Keywords

  • C. elegans
  • Stress response
  • GFP reporter transgene
  • Pesticides
  • Soil organic residues