EnvironOme Integrating Omics in ecotoxicology: tools for environmental risk assessment PTDC/AGR-PRO/3496/2012

Transcription

1 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, T 1,2,3, 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 1 MARE 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;

2 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.

3 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 LABORATORY EFFECT ASSESSMENT PROTEOMICS FIELD EFFECT ASSESSMENT INTEGROMICS

4 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.

5 Methods Survival/Reproduction Tests Reproduction: - Main biological process adressed - Relate Omics to a higher level of organization Folsomia candida ISO guideline 11267: 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 ( ) mg a.i./kg LC50 = >4.32 mg a.i./kg

6 Methods Laboratorial Exposure Tests CT Glyphosate EC50 Day 4 Day 7 Day 10 Replicates per treatment: 6 replicates Omics (Trizol method) 75 organisms per replicate Samples kept at -80⁰C Day 28 5 replicates per treatment to follow reproduction and verify EC50 5 replicates for reproduction 10 organisms per replicate

7 Methods Laboratorial Exposure Tests Survival and Reproduction effects Glyphosate Reproduction and Survival Adults Juveniles Juveniles % Adults Ct EC 50 0

8 Methods Omics Methodology 1. RNA / Protein Extraction Total RNA and proteins isolated with TRIzol Reagent methodology. Homogenization + Chloroform g, 15 min 2. Transcriptomics RNA-Seq TruSeq Library Preparation Kit Illumina HiSeq x 16 x 10 9 bp 2 x 100 bp read length 3. RNA-Seq data assembly using Trinity algorithm

9 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

10 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

11 Results Protein Levels Differential proteins along time-points 4 Days ATP Metabolism Response to heat Cytoskeleton Development 7 Days Oxidation-Reduction Process Response to Stress Protein Synthesis Days Egg Yolk formation Chitin Metabolism Muscle contraction Regulation Body Morphogenesis Regulation

12 Results Gene and Protein Indicators Biological functions Involved Proteins 4 Days ATP Metabolism Response to heat Cytoskeleton Development 7 Days Oxidation-Reduction Process Response to Stress Protein Synthesis 10 Days Egg Yolk formation Chitin Metabolism Muscle contraction Regulation Body Morphogenesis Regulation Genes FA biosynthetic process Oxidation-reduction process Sphingoid metabolic process Vitelline membrane formation Chitin metabolic process Phospholipid biosynthetic process Sphingomyelin catabolic process Proteolysis

13 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 10 Days No significant correlations

14 Results Gene/Protein Correlations Looking to more specific functional groups Enzymes Muscular Proteins Stress Related Proteins (SRP) 4 Days Correlation: 0,327 p-value: 0,244 n: 14 Correlation: 0,119 p-value: 0,749 n: 8 Correlation: 0,524 p-value: 0,16 n: 8 7 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 10 Days S. Correlation: 0,543 p-value: 0,043 n: 14 S. Correlation: 0,762 p-value: 0,02 n: 8 Correlation: 0,952 p-value: 0, n: 8

15 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 Affected lipid metabolism Oxidative Phosphorylation uncoupling Repression of chitin metabolism Lower reproductive rates Cellular Respiration/ Aerobic metabolism affected Moulting and Growth impairment

16 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.

17 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.

18 Acknowledgements Sara Novais Marco Lemos Tiago Natal-da-Luz José Paulo Sousa Tjalf de Boer Dick Roelofs Nico van Straalen Bart Devresse Project: ENVIRONOME-PTDC/AGR-PRO/3496/2012) research grants: SFRH/BD/98266/2013, SFRH/ BPD/94500/2013) and SFRH/BPD/79478/2011