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Metabolite Assignment and Profiling of Environmental Stressors in Earthworms (Eisenia fetida), Coelomic Fluid, and Coelomocytes

  • Author(s): Griffith, Corey Michael
  • Advisor(s): Larive, Cynthia K
  • et al.
Creative Commons 'BY' version 4.0 license
Abstract

Earthworms (Eisenia fetida) are abundant and vital members of the soil environment and monitoring their metabolism may be a useful indicator of soil health and ecotoxicity. This dissertation aimed to expand the analysis of nonlethal and noninvasive earthworm metabolite pools to probe their usefulness in environmental monitoring. Coelomic fluid (CF) is a biofluid that fills the body cavity of the worm and contains free-moving liver-like and immune cells called coelomocytes (CC). 1H NMR, GC-MS, and LC-MS were used to identify metabolites in earthworm, CF, and CC extracts. Fifty-four metabolites were detected in earthworm extracts, 47 in CF, and 41 in CC using 1H NMR. GC-MS was only employed with CF extracts, where 44 metabolites were detected. Targeted LC-MS analyses detected 97 metabolites in earthworm extracts, 82 in CF, and 67 in CC. Significantly, assignment of the earthworm metabolomes led to the identification of a new metabolite: (-)-beta-ʟ-malyl-ʟ-glutamate. Malylglutamate was detected in several invertebrate species at concentrations in the mM - uM range, and it was elucidated as a chelator and potential store for malate and glutamate. This dissertation also sought to explore the response of earthworm metabolism to environmental stressors. CF of earthworms exposed to six chloroacetanilide herbicides (acetochlor, alachlor, butachlor, metolachlor, S-metolachlor, and propachlor) were collected and analyzed using 1H NMR and GC-MS. Perturbations in lipid metabolism and beta-oxidation were observed, suggesting that chloroacetanilide herbicides affected earthworms in a manner similar to the herbicidal mode of action. Exposure to chlorothalonil was used to compare the impact of metabolites in earthworm, CF, and CC extracts using 1H NMR and LC-MS. CF extracts were the most sensitive matrix to detect the effects of chlorothalonil exposure, where increased glutamine levels was the only biomarker detected at both doses. Chemometrics revealed N-acetylserine and ophthalmic acid as strong biomarkers in the high dose group of CF extracts, which may indicate increased oxidative stress. ADP ribose was the only metabolite consistently affected and its increase could be a response to chlorothalonil-induced DNA damage. This work supports metabolic profiling in earthworm and CF extracts to determine which matrix is most sensitive for detecting environmental stress.

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