**2.1.2** *D. magna* **with unique phenotype**

316 Gel Electrophoresis – Advanced Techniques

*Daphnia* related to regulation of developmental genes, the genetic basis of evolutionary

Understanding and predicting how individual organisms respond at the molecular level to environmental change will provide new insight into the evolution of complex biological systems. This insight will lead to the development of new predictive models of hostpathogen interactions, environmental stress and community dynamics as a function of environment and genotype/phenotype (National Science Board, 2000) advancing the field of individualized molecular medicine. As the number of organisms with complete genome sequence increases and technological improvements allow more sequence to be generated at a lower cost, the ability to look at genetic variation in a variety of organisms is greater than ever. However, to understand the role of genetic variation in the context of the natural environment, a model system with two critical components, (1) well-developed molecular resources and (2) a well-understood ecological knowledge base, is essential. Until recently, model systems typically possessed one of these components in depth while the other was nominal or lacking altogether. The recent sequencing of the entire *Daphnia pulex* genome and the establishment of the still growing molecular toolbox (ESTs, genetic map, arrays, etc.)

Organisms respond to environmental change through relatively quick changes in gene expression or through evolutionary response over multiple generations. To better comprehend the effect of gene expression on phenotype, an understanding of genetic variation for gene expression is necessary. A comprehensive understanding of genetic variation is obtained by sampling between and within populations, including individual organisms, directly from their natural environment. The well-documented ecological understanding of *Daphnia* makes the system uniquely suited to this and allows researchers to collect and sample individuals of wild populations directly from their native

Our goal was to demonstrate that it is possible to detect biologically relevant variation in protein expression from an individual *Daphnia*. Using pressure cycling technology (PCT) for sample preparation and two-dimensional gel electrophoresis (2-DE), we have demonstrated that differences in protein expression between individual *Daphnia* with distinct genotypes and exhibiting biologically relevant phenotypic differences are detectable. The ability to detect and analyze individual differences for a large number of proteins represents an important step towards understanding the connection between genotype/phenotype and

Starter cultures of the green algae *Ankistrodesmus falcatus* were obtained from UTEX, The Culture Collection of Algae at The University of Texas (Austin, TX, USA). *A*. *falcatus* was grown in 2 L aerated, air-filtered culture vessels containing GTk media at 25ºC under continuous illumination. GTk contains the following macronutrients, 0.2 mM CaCl2, 2.5 mM KNO3, 0.3 mM MgSO4, 0.4 mM Na2HPO4 and the following micronutrients, 150 µM EDTA,

ecology, and parasite resistance and immunity.

represents the first model system with both components in place.

environment.

the environment.

**2.1.1 Algae cultures** 

**2. Materials and methods 2.1** *Daphnia* **and algae cultures**  *D. magna* starter cultures were obtained from Sachs Systems Aquaculture (St. Augustine, FL, USA). Stabilized cultures were maintained in 8 L of 25% mineralized water (Vermont Spring Water Company, Brattleboro, VT, USA) at a density of 60-120 individuals/L. *D. magna* were cultured at 22º ± 1ºC under constant illumination with standard fluorescent bulbs. Cultures were maintained at pH 7.0-7.4 by the addition of 100 g/L crushed coral (Tideline Aquatics, Hanahan, SC, USA) supplied in nylon bags. Starter cultures were fed daily with 1 mL/L of *Nanochloropsis* microalgae liquid concentrate (Reed Maricultures, Campbell, CA, USA) for the first four weeks, followed by 0.1 mL/L thereafter.

#### **2.1.3** *D. magna* **with unique genotype and** *D. pulex*

*D. magna* clones Iinb1 and Xinb3 were isolated from Munich, Germany and Tvärminne, Finland, respectively (Rottu et al., 2010). *D*. *pulex* clone Log50 was obtained from the *Daphnia* Genomics Consortium stock (www.wfleabase.org/stocks). Xinb3 and Log50 are the clones for the respective, *D. magna* and *D. pulex* genome projects. Cultures were maintained in 8 L of COMBO media (Killham et al., 1998) at a density of 30 individuals/L at 20° + 1°C under a 16:8 hours, light:dark, low intensity photoperiod, and fed 1mg Carbon/L of *A*. *falcatus*.
