**1. Introduction**

Analysis of individual genetic variation is paramount to understanding how organisms and communities respond to changes in the environment and requires a model system with well-developed molecular resources and a solid foundation of ecological knowledge. Traditional genetic model systems (*E. coli*, yeast, fly, worm, and mouse) have served as workhorses in elucidating virtually all of the knowledge in modern molecular biology. While these systems were chosen for their robustness in laboratory studies, virtually nothing is known about their life histories in their native environment. By contrast, newmodel systems, which have typically been studied in depth, from an ecological perspective are severely limited in regards to their molecular resources.

The model organism *Daphnia* has been utilized as an ecological model for centuries, and now with the sequencing of the genome complete and the development of the associated molecular resources, it is poised as one of the few model systems with the necessary molecular and ecological tools to answer the questions of response to the environment (Colborne et al., 2011). Long recognized as a model for ecological research, the freshwater crustacean *Daphnia* is rapidly maturing into a powerful model for understanding basic biological processes, within an ecological context. A common resident of lakes and ponds, *Daphnia* has been the subject of over a century of study in the areas of rapid environmental response, physiology, nutrition, predation, parasitology, toxicology and behavior (Edmondson, 1987). The reproductive cycle of *Daphnia* is ideal for experimental genetics. Generation time in the laboratory rivals that of almost all other model eukaryotic systems, reaching maturity within 5-10 days. Under favorable environmental conditions, *Daphnia*  reproduce through parthenogenesis, allowing the conservation of genetic lines. Sexual reproduction is induced by environmental changes allowing the production of inbred or outbred lineages. The sexually produced diapausing eggs, termed ephippia, can be stored viably for considerable periods. Moreover, they have been hatched from lake sediments up to a century old (Hairston et al., 2001; Limburg & Weider, 2002) allowing tracking of genetic changes over ecological and evolutionary time scales. *Daphnia* are transparent throughout life, allowing for studies of tissue-specific gene expression at any life stage and direct observation of parasites and pathogens. As a result, there is a growing body of work in

Two-Dimensional Gel Electrophoresis Reveals

(Leland Jahnke, Personal Communication).

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

the first four weeks, followed by 0.1 mL/L thereafter.

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

**2.2 Harvesting of** *Daphnia*

when dehydrated (*n* = 64).

**2.3 Pressure Cycling Technology (PCT)** 

with and without ephippia and from single *Daphnia pulex*.

Differential Protein Expression Between Individual *Daphnia* 317

Na2, 20 µM FeSO4, 2 µM ZnSO4, 1 µM NaMoO3, 0.6 µM CuSO4, CoCl2 and 14 µM MnCl2

*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

*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

*Daphnia* gut contents were minimized by allowing the microcrustaceans to feed on copolymer microspheres of 4.3-micron mean diameter (Duke Scientific, Fremont, CA, USA) for one hour prior to harvesting. Microspheres were fed at a concentration equal to the number of algal cells previously supplied. *Daphnia* were harvested by filtration through 250 um Nitex mesh (Sefar America, Depew, NY, USA) and flash frozen. Average mass of adult *Daphnia pulex* was 0.1158 + 8.3 mg fully hydrated and 0.05285 + 10.60 mg dehydrated (*n* = 50). Average mass of adult *D. magna* was 1.37+ 0.46 mg fully hydrated and 0.23 + 0.06 mg

PCT has been shown to be an effective means for isolating proteins from a variety of microorganisms, as well as many difficult-to-lyse samples such as *Caenorhabditis elegans* (Geiser et al., 2002; Smejkal et al., 2006b; Smejkal et al., 2007). PCT, which subjects samples to rapid cycles of pressure, facilitated the extraction of proteins from single *Daphnia magna*

*Daphnia* were transferred to tared PULSE Tubes (Pressure BioSciences, Inc., South Easton, MA, USA) and suspended in 500 uL of 7M urea, 2M thiourea, and 4% CHAPS supplemented with 100 mM dithiothreitol (DTT) and protease inhibitor cocktail P-2714 (Sigma Aldrich Chemicals, St. Louis, MO). An additional 900 uL of mineral oil was added to accommodate the necessary volume for the PULSE Tubes. The tubes were placed in the Barocycler NEP-3229 (Pressure BioSciences, Inc., South Easton, MA, USA) for 60 pressure

hours, light:dark, low intensity photoperiod, and fed 1mg Carbon/L of *A*. *falcatus*.

*Daphnia* related to regulation of developmental genes, the genetic basis of evolutionary ecology, and parasite resistance and immunity.

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.) represents the first model system with both components in place.

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 environment.

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 the environment.
