Download Using an integrative OMICs approach to unravel Glyphosate

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