Daphnia
Molecular and Population Stress Responses in Daphnia magna
Microarray gene expression profiling is a powerful tool for defining holistically the effects of environmental change on biological function. This new technology has many potential applications in the field of ecotoxicology. However, as the science of ecotoxicology evolves, it is becoming increasingly apparent that stronger links must be forged between the molecular and the population responses to stressors. Our research aims to strengthen these links by comparing the effects of different stressors on gene expression (using microarrays) with effects observed at the population level (using image analysis) in the widely used indicator species Daphnia magna.
Stressors, which include toxicants and physico-chemical aspects of an organism's environment, (e.g. pH, water hardness) may influence individual fitness by reducing somatic growth, fecundity and survival. Together these life history traits determine a population's growth rate (PGR), which may be positive, zero, or negative in declining populations. Since stress responses are often preceded by alterations in gene expression, microarray studies offer insights into the overall health of individuals and consequently, that of a population. For example, genes up- or down-regulated in response to acute stress may predict chronic effects at the population level. By linking changes in gene expression to changes in PGR we hope to provide a fundamental understanding of the nature of stress responses in Daphnia magna.
Personnel
- Professor Richard Sibly
- Dr Amanda Callaghan
- Dr Richard Connon
- Lars-Henrik Heckmann
Collaborators
- Tom Hutchinson AstraZeneca Brixham
- Steve Maund – Syngenta, BASEL, Jonathan Moggs - Syngenta, Central Toxicology Laboratories
Past Presentations
- NC3Rs Westminster poster 2007 (PDF-470KB)
- SET for BRITAIN Westminster poster 2007 (PDF-196KB)
- SETAC Hamburg poster 2003 (PDF-1.6MB)
- British Toxicology Society - Edinburgh 2004 poster (PDF-8MB)
- SETAC Lille poster 2005 (PDF-140KB)
- SETAC The Hague poster 2006 (PDF-671KB)
Standard Operation Procedures
- Culturing Daphnia magna (PDF-201KB)
- Culturing Chlorella vulgaris (PDF-156KB)
- Daphnia Digital Image Analysis (PDF-152KB)
- GeneSpring Analysis (PDF-97KB)
Links
Gene Expression Studies in Daphnia magna using Microarrays
We have developed a microarray with over 12000 gene fragments. These fragments consist of a cDNA library of unexposed D. magna*, suppressive subtractive hybridisation (SSH) between adults and juveniles**, and SSHon populations exposed to selected stressors: cadmium, lufenuron, pH, hardness, kerosene and ibuprofen***.
Approximately 250 third brood neonates (<48h old) per treatment were used to obtain sufficient total RNA. A reference RNA pool was prepared from over 10,000 D. magna (<48 h old).cDNA probes labelled with fluorescent CY3 or CY5 dyes (Amersham Pharmacia, UK) were generated using 10-20 mg total RNA.Microarrays were scanned using a GenePix 4000b scanner (Axon) to quantitate hybridisation fluorescence and results were analysed using GeneSpring (Silicon Genetics).
*Kindly donated by Dr Hajime Watanabe and Dr Taisen Iguchi - Center for Integrative Bioscience, Okazaki National Research Institutes, Japan.
**Kindly donated by Dr Wim de Coen – Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Belgium.
***Carried out at by our research team at the University of Reading.
Population Studies in Daphnia magna using Image Analysis
We use a rapid, non-destructive and validated digital imaging system to estimate population density and population surface area to calculate population growth rate. Populations are maintained in static, glass systems and changes in population growth or decline are recorded using high-resolution digital photography. Image Analysis software is used to transform the colour images to a binary image before automatically quantifying the number and the surface area of the selected white areas of the image.