MONITORING POLLUTION IN MARINE & FRESHWATER USING MUSSELS, THREE SPINED STICKLEBACK & qPCR Kneller...

MONITORING POLLUTION IN MARINE & FRESHWATER USING

MUSSELS, THREE SPINED STICKLEBACK & qPCR

Kneller C, Baker PJ, Miller B Redshaw J and Craft JA

OUTLINE

What is Ecotoxicology? Why Environmental Genomics? Genomics Methods Project Results Summary

ECOTOXICOLOGY

• study of how biological systems respond to stress caused by environmental contaminants.

• incorporates aspects of ecology, toxicology, physiology, molecular biology, analytical chemistry and many other disciplines

• ultimate goal is to be able to predict the effects of pollution.

• so the most efficient and effective action to prevent or remediate any detrimental effect can be identified.

What is Ecotoxicology?

Why Environmental Genomics?

Genomics Methods

Project

Results

Summary

ENVIRONMENTAL GENOMICS

SEPA use standardised tests that produce values that can indicate if an organism has been affected by an external factor.

results of such tests range from effect to lethality on the organism as a whole.

21st Century = knowledge of DNA Why not identify genes that have shown to be

‘stressed’ by certain environmental factors? Use them to monitor responses and effects of

pollutants and stressors on organisms.



Genomics Methods

Project

Results

Summary

mRNA



Genomics Methods

Project

Results

Summary

mRNA = A molecule that encodes for Proteins

PCR-Polymerase Chain Reaction



Genomics Methods

Project

Results

Summary

What is PCR? •a technique that amplifies a single or few copies of a piece of DNA•mRNA = fragile and easily broken down – Convert to cDNA.

qPCR



Genomics Methods

Project

Results

Summary

•qPCR = Quantitative PCR or Real-Time PCR

•Uses a dye that fluoresces in presence of DNA

•Measured by Real-Time PCR Machine.

Basically the results from the graph enables us to monitor changes in the expression of genes in each sample.

MONITORING POLLUTION IN MARINE & FRESHWATER USING MUSSELS, THREE

SPINED STICKLEBACK & qPCR Freshwater = Three-spined Stickleback,

Gasterosteus aculeatus as a Sentinel Species.

Marine = Mussel, Mytilus Edulis as a Sentinel Species.



Genomics Methods

Project

Results

Summary

Genes of Interest?



Genomics Methods

Project

Results

Summary

•Sewage (Oestradiol)•Metals•Hydrocarbons

Identified various genes of each type within both Stickleback and Mussels.

Using qPCR tried to establish if we could use these genes as ‘stress indicators’ when exposed to the contaminants of interest.

Did we succeed?

Choriogenin H & L- Males Lugar Water

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BLANK EWC LUG 2-D-M

LUG 3-D-M

LUG 5-D-M

LUG 7-D-M

LUG 53-U-M

LUG 25-U-M

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xpre

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BLANK EWC LUG 2-D-M

LUG 3-D-M

LUG 5-D-M

LUG 7-D-M

LUG 25-U-M

LUG 53-U-M

Cho

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sion

Downstream

Upstream

Ch H Ch LWhat is Ecotoxicology?


Genomics Methods

Project

Results

Summary

Metallothionien Lugar Water Females Up/Down stream

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EWC LUG 3-D LUG 5-D LUG 53-D LUG 9-D LUG 1-D LUG 25-U LUG 27-U LUG 30-U LUG 70-U LUG 73-U LUG 2-D

Met

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thio

nien

Exp

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E-W

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Downstream Upstream



Genomics Methods

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Summary

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Catalase Expression relative to Gill calibratorMT20II Expression relative to Gill calibrator

Woodhall Garnock Colintraive

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MT20II

Catalase

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Gill : Catalase & MT20II



Genomics Methods

Project

Results

Summary

Genes:VDG3

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VDG3 Expression relative to Mantle Calibrator


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VDG3 Expression in digestive gland


Tissue-Mantle

Tissue-Digestive gland

Mean +/-SEM

020406080

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VDG3 Digestive Gland

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VDG3 MantleWhat is Ecotoxicology?


Genomics Methods

Project

Results

Summary

Summary

Shown that gene expression can be measured in polluted sites.

Genes that WERE induced matched the contamination present.

Potential to infer pollution from the levels of the genes measured.

Further Work• Look for more genes within mussels.• Increase in numbers of mussels tested.• Potential to apply to other organisms e.g. earthworms within soils



Genomics Methods

Project

Results

Summary

MONITORING POLLUTION IN MARINE & FRESHWATER USING MUSSELS, THREE SPINED STICKLEBACK & qPCR Kneller...

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