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EnvironOme Integrating Omics in ecotoxicology: tools for environmental risk assessment PTDC/AGR-PRO/3496/2012 Using an integrative OMICs approach to unravel Glyphosate mechanisms of toxicity in Folsomia candida Simões, T1,2,3, Novais, S.C.1,3, Natal-da-Luz, T.2, Sousa, J.P.2, Devreese, B.4, de Boer, T.3, Roelofs, D.3, van Straalen, N.3, Lemos, M.F.L.1 – Marine and Environmental Sciences Centre, ESTM, Polytechnic Institute of Leira, Peniche, Portugal; 2 Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Portugal; 3 Institute of Ecological Sciences, Vrije University, Amsterdam, Netherlands; 4 Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, Belgium; 1 MARE Introduction State of the Art / Project Context Complementary information to the elucidation of mechanisms of toxic action Omics have been used in Ecotoxicology as fast detection tools. Need to validate reliable tools (Omics) in more realistic field scenarios. Introduction Main Focusing Point Can protein and gene differential expression be used as reliable tools to help predicting effects of pesticides in soil invertebrates under more realistic contamination scenarios? Folsomia candida SURVIVAL/REPRODUCTION TRANSCRIPTOMICS FIELD EFFECT ASSESSMENT LABORATORY EFFECT ASSESSMENT PROTEOMICS INTEGROMICS Methods Test soil and Pesticide Formulation Agricultural Natural Soil Low Mondego Region, Coimbra, Portugal Kept in fallow for 10 years Montana® (30.8 % a.i. Glyphosate) Sold since 1974 as a broad-spectrum herbicide. One of the most commonly used herbicides worldwide. Mode of action: - Aromatic amino acid synthesis enzymatic inhibiton/plant growth regulator. - According to some studies it may cause uncoupling of oxidative phosphorylation and endocrine disruption. Methods Survival/Reproduction Tests Reproduction: - Main biological process adressed - Relate Omics to a higher level of organization Folsomia candida • ISO guideline 11267:1999 • • • • • 5 concentrations + control 5 replicates 30g soil/replicate 10 org (10-12 days old) /replicate (Synchronized cultures) 28 days – 20⁰C; 16:8 h (light:dark) 10 Control EC50 = 4.95 (0.99 – 8.90) mg a.i./kg LC50 = >4.32 mg a.i./kg Methods Laboratorial Exposure Tests Glyphosate CT EC50 Day 4 Replicates per treatment: Day 7 • • • 6 replicates Omics (Trizol method) 75 organisms per replicate Samples kept at -80⁰C • • 5 replicates for reproduction 10 organisms per replicate Day 10 Day 28 5 replicates per treatment to follow reproduction and verify EC50 Laboratorial Exposure Tests Survival and Reproduction effects Glyphosate Reproduction and Survival 1000 12 Adults Juveniles 800 10 8 600 25% 6 400 4 200 2 0 0 Ct EC 50 Adults Juveniles Methods Methods Omics Methodology 1. RNA / Protein Extraction Total RNA and proteins isolated with TRIzol® Reagent methodology. Homogenization + Chloroform + 12000g, 15 min 2. Transcriptomics RNA-Seq TruSeq Library Preparation Kit Illumina HiSeq 2000 8 x 16 x 109 bp 2 x 100 bp read length 3. RNA-Seq data assembly using Trinity algorithm Methods Omics Methodology 4. Proteomics iTRAQ 8-plex Methodology (relative and absolute quantitation): Samples underwent a 2D Fractionation LC-MS/MS Analysis (4800 Plus MALDI TOF/TOF) Protein Identification/quantification with ProteinPilot Software Results Gene Expression Late Response 25% reduction on reproduction Gene Ontology (GO) enrichment analysis FA biosynthetic process Oxidation-reduction process Vitelline membrane formation Sphingoid metabolic process Chitin metabolic process Phospholipid biosynthetic process Sphingomyelin catabolic process Proteolysis Results Protein Levels Differential proteins along time-points 4 Days 7 Days Oxidation-Reduction Process Response to Stress Protein Synthesis ATP Metabolism Response to heat Cytoskeleton Development 29 10 Days Egg Yolk formation Chitin Metabolism Muscle contraction Regulation Body Morphogenesis Regulation Results Gene and Protein Indicators Biological functions Involved Proteins 4 Days 7 Days 10 Days ATP Metabolism Response to heat Cytoskeleton Development Oxidation-Reduction Process Response to Stress Protein Synthesis Egg Yolk formation Chitin Metabolism Muscle contraction Regulation Body Morphogenesis Regulation Oxidation-reduction process Vitelline membrane formation Sphingoid metabolic process Chitin metabolic process Genes FA biosynthetic process Phospholipid biosynthetic process Sphingomyelin catabolic process Proteolysis Results Gene/Protein Correlations Gene to Protein individual correlations However… -Genes coding for the significant proteins were not differentially expressed in the RNAseq experiments. Therefore, -List of all identified proteins and respective expressed coding genes were used to test the correlations. 4 Days 7 Days No significant correlations… 10 Days Results Gene/Protein Correlations Looking to more specific functional groups Enzymes Correlation: 0,327 p-value: 0,244 n: 14 Muscular Proteins Correlation: 0,119 p-value: 0,749 n: 8 Stress Related Proteins (SRP) Correlation: 0,524 p-value: 0,16 n: 8 4 Days Correlation: -0,06 p-value: 0,820 n: 14 Correlation: -0,286 p-value: 0,460 n: 8 Correlation: -0,643 p-value: 0,07 n: 8 7 Days S. Correlation: 0,543 p-value: 0,043 n: 14 10 Days S. Correlation: 0,762 p-value: 0,02 n: 8 Correlation: 0,952 p-value: 0,0000002 n: 8 Discussion Stronger molecular response after 10 days. - Higher response before 4 days? - Response associated with 25% effect on reproduction. Less significant protein levels than differentially expressed genes. - Few Identified Proteins - Low Proteomics data associated to the technique limitations? Proposed mechanisms of glyphosate toxic action in F. candida: Impairment in egg yolk and vitelline membrane formation Lower reproductive rates Affected lipid metabolism Oxidative Phosphorylation uncoupling Repression of chitin metabolism Cellular Respiration/ Aerobic metabolism affected Moulting and Growth impairment Discussion Best correlations between expression of genes and proteins were found for the same time point samples. Higher correlations in samples of 10 days exposure Less biological variability Specific sets of Genes/Proteins exhibited higher correlations. Although individual correlations were weak, the general affected functions presented good similarity between Omics datasets. Conclusions Challenge to link effects at different levels of biological organization. First attempt to understand the mechanisms of toxicity behind the effects of glyphosate in F. candida. Integrated Omics approach can help to unravel pesticide modes of action. Provide useful insights that may not be deciphered from individual analysis of gene or protein expressions. Acknowledgements Sara Novais Tjalf de Boer Marco Lemos Dick Roelofs Project: ENVIRONOME-PTDC/AGR-PRO/3496/2012) research grants: SFRH/BD/98266/2013, SFRH/ BPD/94500/2013) and SFRH/BPD/79478/2011 Nico van Straalen Tiago Natal-da-Luz José Paulo Sousa Bart Devresse