Two-Dimensional Gel Electrophoresis Reveals Differential Protein Expression Between Individual Daphnia

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.


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 2.1.3D. 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.

Harvesting of Daphnia
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 when dehydrated (n = 64).

Pressure Cycling Technology (PCT)
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 with and without ephippia and from single Daphnia pulex.
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 www.intechopen.comGel Electrophoresis -Advanced Techniques 318 cycles, each cycle consisting of 10 seconds at 35,000 psi followed by rapid depressurization and held for 2 seconds at atmospheric pressure.Following PCT, each PULSE Tube was coupled to an Ultrafree-CL centrifugal filtration device with a 5-micron pore size (Millipore Corporation, Danvers, MA, USA) and evacuated by centrifugation for 1 minute at 1000 RCF.The PULSE Tube was removed and centrifugation continued for 4 minutes at 4000 RCF, followed by the removal of the mineral oil.

Reduction, alkylation, and ultrafiltration
Samples were transferred to ULTRA-4 ultrafiltration devices with 10 kDa molecular weight cut-off (Millipore Corporation, Danvers, MA, USA).Proteins larger than 10 kDa are retained in the ultrafiltration device.Centrifugation assisted the ultrafiltration, and the samples were exchanged with fresh UTC until the final DTT concentration was 10 mM.Reduction and alkylation of the samples were performed directly in the ultrafiltration devices using 5 mM tributylphosphine and 50 mM acrylamide as described (Smejkal et al., 2006a).The alkylation reaction was terminated by resuming centrifugation and ultrafiltrative exchange.Bradford Reagent (Sigma-Aldrich Chemicals, St. Louis, MO, USA) was used to measure the protein concentration in each sample.

IEF and 2-DE
Two-hundred uL of each sample was placed onto individual wells in IPG rehydration trays from Proteome Systems (Woburn, MA, USA).Bio-Rad ReadyStrip IPG strips with a pH range of 3-10, 4-7, or 7-10 (Hercules, CA, USA) were placed onto each sample, and the tray was placed into a humidifying Ziploc bag.Rehydration occurred over six hours until all the sample was visibly absorbed by the strip.At the termination of rehydration, strips were placed into isoelectric focusing trays and ran at 10,000 volts (maximum voltage) for 110,000 accumulative volt-hours.Strips were equilibrated twice for 10 minutes in 375 mM Tris-HCl containing 2.5% SDS, 3M urea, 0.01% and phenol red, then placed onto Criterion Tris-HCl 8-16% IPG+1 gels (Bio-Rad Laboratories, Hercules, CA) and ran at 120 V and 60 mA/gel.2-DE gels were ran for all IPGs, only 4-7 gels are shown.

Digital image analyses
The 24-bit images were analyzed using PDQuest™ software (Bio-Rad, v.7.1).Background was subtracted, and protein spot density peaks were detected and counted.A reference pattern was constructed from one of the individual gels to which each of the gels in the matchset was matched.Numerous proteins that were uniformly expressed in all patterns were used as landmarks to facilitate rapid gel matching.After matching, the total spot count was determined for each gel.

Protein variation between individual D. magna of distinct genotypes
Our first goal was to demonstrate that differences in protein expression could be detected between individual Daphnia of distinct genotypes.Individual D. magna from Iinb1 and Xinb3 genotypes were isolated, proteins extracted and analyzed in quadruplicate by 2-DE as www.intechopen.comTwo-Dimensional Gel Electrophoresis Reveals Differential Protein Expression Between Individual Daphnia 319 describe above.Silver staining detected an average of 687 + 11 protein spots from the Xinb3 gels and 692 + 14 protein spots from the Iinb1 gels (figure 1).After normalization of the gel images based on total intensity, 679 spots were matched between the two gel images.One Hundred thirty six spots showed a two-fold or greater difference in spot intensity.Seventynine of these were more abundant in Xinb3 and 57 were more abundant in the Iinb1.To illustrate these differences, the silver stained gels were digitally colored.The Iinb1 gels were red and the Xinb3 gels were green.The gels were then superimposed.(Figure 2).

Protein variation between individual D. magna of distinct phenotypes
We were also able to detect differences between individual Daphnia with distinct phenotypic differences.Individual D. magna, with and without ephippia, were isolated, proteins extracted and analyzed by 2-DE as described above.Silver staining detected an average of 524.5 ± 7.8 protein spots in 2D gels produced from single D. magna, with and without ephippia (figure 3).After normalization of the gel images based on total intensity, 386 spots were matched between the two gel images.Eighty-four spots showed a three-fold or greater difference in spot intensity.Fifty-five of these were more abundant in the parthenogenic (no ephippia) animal, while 29 were more abundant in the sexual animal.In addition, eleven protein spots were unique to the parthenogenic phenotype, while 49 protein spots were unique to the sexual phenotype.This demonstrates the feasibility of 2-DE and image analysis for the differentiation of Daphnia phenotypes isolated in the field as indicators of environmental variables.Other studies with parthenogenic and sexual Daphnia carinata were able to identify several proteins that were differentially expressed between the two phenotypes by 2-DE; however, 100's of animals were used (Zhang et al., 2006).It is interesting to note that using single animals, we discovered similar patterns of up-regulation in the parthenogenic individual in comparison to the sexual phenotype.While Zhang et al.'s goal was only to gain insight into the genes involved in the switch to sexual reproduction, our method, using single Daphnia magna with and without ephippia, allows the sampling of these candidate genes within a population.

Protein functional diversity
To properly evaluate our method, it is necessary to understand the functional diversity of the proteins sampled.The proteins detected on any 2D gel will be biased towards the most abundant and the goal is to sample gene expression for proteins of diverse functions.To evaluate the likely diversity of proteins detectable and comparable by this method, we first generated a theoretical 2D gel for the 2000 most highly expressed genes in the Daphnia pulex genome.The correlation between mRNA expression levels and protein abundance is a debated topic; (for a brief review, see Greenbaum et al., 2003) however, recent studies have found a high correlation (Lu et al., 2007;Tuller et al., 2007).Using the recently completed draft of the Daphnia pulex genome, we found the top 2000 gene prediction models with the most Expressed Sequence Tag (EST) evidence using BLAST (Altschul et al., 1990).The theoretical pI and MW of these genes were calculated using the Compute pI/MW tool from ExPASy (Gasteiger et al., 2003) and the results graphed using Excel to create the theoretical 2D gel (Figure 6).Importantly, the pI range used on the actual gels (indicated by the rectangle) covers a significant portion of the predicted proteome.
To assay the functional diversity of these theoretical proteins, we utilized the 25 eukaryotic orthologous groups (KOGs) (Totusov et al., 2003) that were assigned to the Daphnia pulex genes as part of the genome sequence annotation project.Many D. pulex genes have no homology to any entries at NCBI; therefore, it is not surprising that of the approximately 30,000 predicted genes only 18,371 have been assigned to a KOG class (Colbourne et al., 2011) Of the 2000 most highly expressed genes, 298 had been assigned to a KOG class.Twenty four of the 25 KOG classes were represented, and only "coenzyme transport and metabolism" was absent.To understand the functional diversity that may be present on a single animal 2D gel of approximately 1000 spots, we looked at the KOG assignments of the 1000 most highly expressed genes.Ninety-seven have been assigned to a KOG class, with 21 of the 25 KOG classes being represented.Not represented were "coenzyme transport and metabolism", "secondary metabolites, biosynthesis, transport and catabolism", "nuclear structure" and "chromatin structure and dynamics" (Figure 7).In general, KOG classes that are well represented in the Daphnia genome are also well represented in the top 2000 of most highly expressed genes.As 84% of the KOG classes are represented in the top 1000 most highly expressed genes, we feel that a single animal 2D gel of approximately 1000 spots should represent a diverse sampling of biologically relevant proteins.(18,371) assigned to a KOG class (Right-hand axis).To further characterize the predicted protein gel, we compared it to our observed spot counts.Table 1 summarizes the number of predicted proteins and the number of actual proteins within specific pI ranges.Through the generation of several D. magna (both single animal and multiple-animal) 2D gels (not shown), we were able to visualize a total of 1285 protein spots.Seventy percent of these were observed in the 4-7 pI range, while the theoretical gel predicted 51% in this same range.It is important to note that the theoretical 2D does not account for post-translational modifications and was generated from D. pulex genes.Both of these factors will contribute to differences between the predicted (D. pulex) and observed (D. magna) number of proteins.

Basic proteins constituents of the D. magna proteome
Initially, single Daphnid extracts were analyzed on IPG strips with a pH range of 3-10.Since more than 80% of the proteins visualized by silver staining were in the pH 4-7 range, subsequent analyses were performed using IPGs pH 4-7.However, the theoretical 2D does predict a significant number of proteins (47%) in the basic range (7-10).Due to their relative low abundance, the visualization of very basic proteins (pH 7-10) in Daphnids required many more organisms.For this, 184 ± 3 mg of D. magna (approximately 135 organisms) were cultured under either normal or hypoxic conditions and processed in 1.3 mL of ProteoSOLVE IEF Reagent.The samples were concentrated two-fold by ultrafiltration, and IEF was performed on IPGs pH 7-10, followed by silver staining and image analysis.Silver staining detected 355 and 408 spots (gels not shown) in pH range 7-10 from D. magna cultured under normal or hypoxic conditions respectively, further illustrating the utility of 2-DE for detecting phenotypic differences influenced by specific environments.

Conclusion
Organisms live in ever changing environments.Understanding how individuals respond and adapt to these environments on a molecular level forms the basis for advances in personalized medicine and requires model systems with both well-developed ecological knowledge and molecular resources.The freshwater crustacean Daphnia now has these two requirements in place.We have demonstrated the ability of 2-DE to identify protein differences between single Daphnia magna with distinct genotypes (Iinb1 and Xinb3), distinct phenotypic differences (presence or absence of ephippia) and when cultured in different environments (normal or hypoxic conditions).
We predict that the detectable proteins on a single animal 2D gel, while biased towards the most abundant proteins, represent a functionally diverse set of proteins.This technique represents an important step to a greater understanding of individual variation of gene expression and is critical to advancing the field of EEFG.However, as the use of silver stain convolutes downstream mass spectrometry, the number of protein spots that can be confidently identified is significantly curtailed.The full potential of single animal gels will be realized with the development of comprehensive 2D maps of the Daphnia proteome.

Fig. 2 .
Fig. 2. Digitally enhanced, superimposed, silver-stained 2D gels of individual Daphnia magna with distinct genotypes.Red indicates spots unique to Iinb1, green indicates spots unique to Xinb3, yellow indicate spots shared by both genotypes.
Figure 5   shows the number of protein spots detected and the standard deviation for Daphnia pulex gels of 1, 2, 3, 4 and 5 individuals.The low standard deviation indicates that PCT and 2-DE is an efficient and highly reproducible method of sample preparation and protein comparison.

Fig. 5 .
Fig.5.Graph of detected protein spots from duplicate silver stained 2D gels of proteins extracted 1, 2, 3, 4 & 5 Daphnia pulex.The shape of the curve suggests that our method is an efficient and reproducible approach to protein extraction and the majority of the unique proteins are recovered from a single animal.

Fig. 7 .
Fig. 7. Distribution of highly expressed D. pulex genes across 25 KOG classes.Blue and red bars indicate the number of genes in each class from the top 2000 and 1000, respectively, most highly expressed genes (Left-hand axis).Green bars indicate the total genes assigned to each KOG class as a percentage of the total number of genes(18,371)  assigned to a KOG class (Right-hand axis).To further characterize the predicted protein gel, we compared it to our observed spot counts.Table1summarizes the number of predicted proteins and the number of actual proteins within specific pI ranges.Through the generation of several D. magna (both single animal and multiple-animal) 2D gels (not shown), we were able to visualize a total of 1285 protein spots.Seventy percent of these were observed in the 4-7 pI range, while the theoretical gel predicted 51% in this same range.It is important to note that the theoretical 2D does not account for post-translational modifications and was generated from D. pulex genes.Both of these factors will contribute to differences between the predicted (D. pulex) and observed (D. magna) number of proteins.

Table 1 .
Distribution of 2000 most abundant Daphnia proteins predicted from genome sequence