NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Write or delete: DATE GENEPEASE From...

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTEAARHUS UNIVERSITY Write or delete: DATE

GENEPEASE

From ecotoxicogenomics to a soil ecological model system

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTEAARHUS UNIVERSITY Jan. 7, 2011Paul Henning Krogh

Lars Henrik Heckmann Jesper Givskov Sørensen

GENEPEASE collembolan studies› WP 2.2 Transcript response to stress in F.

candida

› WP2.2.1 Microarrays

› WP2.2.2 temporal qPCR of selected genes at EC10 and EC50 effect levels

› WP3 Transcripts in a long-term mesocosm field model system



Microarray and qPCR of a collembolan

› iSQ Chip Folsomia candida Collembola VU version 1.0 for gene expression analysis (Dick Roelofs, VU Amsterdam)

› Microarray overview of the genes that are differentially expressed in control and pesticide exposed F. candida

› The temporal expression of selected target genes will be monitored, using real-time quantitative PCR (qPCR),

› Selected target genes should preferably respond to many pesticides



Transcriptomic responses in Folsomia candida – potential outcome

› ≈ 100 transcripts may be differential in response to the EC10

and EC50

› Upregulation of transcripts encoding for xenobiotic

detoxification and biotransformation enzymes (phases I, II,

and III)

› General stress response

› Oxidative stress – antioxidant transcripts

› Chitin metabolism

› Suppression of protein translation (allocating energy to

detoxification!)

› Immune responses

Nota, B., Bosse, M., Ylstra, B., van Straalen, N.M., Roelofs, D., 2009. Transcriptomics reveals extensive inducible biotransformation in the soil-dwelling invertebrate Folsomia candida exposed to phenanthrene. BMC Genomics 10:236.

Yuan, Y., Ke, X., Chen, F., Krogh, P.H., Ge, F., 2011. Decrease in catalase activity of Folsomia candida fed a Bt rice diet. Subm. to Enviromental Pollution.



What is a microarray?

Microarrays provide a global overview of the genes that are induced/suppressed in a model organism in a specific biological context



How does a microarray work?

Exposed F. candida



Real-time quantitative PCR (QPCR)

To estimate the initial quantity of a specific DNA sequence (target gene)



Principles of QPCR



Principles of QPCR

Baseline (threshold)

Cycle threshold (Ct)

3 cycles difference between green and blue sample= 23 = 8 fold difference



Detoxification phases› In phase I

toxic compounds are modified resulting in more reactive

› metabolites (cytochrome P450s)

› phase IIreactive metabolites are conjugated by transferases with chemical groups such as glutathione or glucuronic acid

› phase III specialized transporters recognize the conjugates, and expel them out of the cell

Nota et al. 2009

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