Angel P. Diz

Proteomics in evolutionary ecology: linking the genotype with the phenotype

The study of the proteome (proteomics), which includes the dynamics of protein expression, regulation, interactions and its function, has played a less prominent role in evolutionary and ecological investigations in comparison with the study of the genome and transcriptome. There are, however, a number of arguments suggesting that this situation should change. First, the proteome is closer to the phenotype than the genome or the transcriptome, and as such may be more directly responsive to natural selection, and thus closely linked to adaptation. Second, there is evidence of a low correlation between protein and transcript expression levels across genes in many different organisms. Finally, there have been some recent important technological improvements in proteomics methods that make them feasible, practical and useful to address a wide range of evolutionary questions even in nonmodel organisms. The different proteomic methods, their limitations and problems when interpreting empirical data are described and discussed. In addition, the proteomic literature pertaining to evolutionary ecology is reviewed with examples, and potential applications of proteomics in a variety of evolutionary contexts are outlined. New proteomic research trends such as the study of posttranslational modifications and protein–protein interactions, as well as the combined use of the different ‐omics approaches, are discussed in relation to the development of a more functional and integrated perspective, needed for achieving a more comprehensive knowledge of evolutionary change.

The consequences of sample pooling in proteomics: An empirical study

Pooling of samples in proteomics experiments might help overcome resource constraints when many individuals are analysed. The measured biological variation should be reduced giving increased power to detect treatment differences. Pooling has been advocated in microarray work but there are few tests of its potential in proteomics. In this study, we examine three issues on which the success of the pooling approach might hinge and provide evidence that: (i) the protein expression in a pool matches the mean expression of the individuals making up the pool for the majority of proteins, although for some proteins the pool expression is different; (ii) the biological variance between pools is reduced compared with that between individuals, as predicted in theory, but this reduction is not as large as expected. A practical consequence of this is that power could be reduced; (iii) proteins detectable in individual samples are usually but not always visible when samples are pooled. We conclude that pooling of samples in proteomics work is a valid and potentially valuable procedure but consideration should be given to these issues in experimental design.

Multiple Hypothesis Testing in Proteomics: A Strategy for Experimental Work

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis. We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Polymorphic microsatellite markers for blue mussels (Mytilus spp.)

Blue mussels of the genus Mytilus (M. edulis Linnaeus 1758; M. galloprovincialis Lamarck 1819; M. trossulus Gould 1850) are widely distributed in Southern and Northern hemispheres. This ecological plasticity together with the existence of interspecific hybridization in overlapping regions (Skibinski et al. 1978) makes them an interesting model for studies of population dynamics in marine habitats. Genetic marker surveys on Mytilus spp. have shown the genetic uniqueness of each species (McDonald and Koehn 1988). Several molecular markers have been developed over the last three decades to describe genetic polymorphisms in mussels. However most of them have a number of disadvantages for use as population markers, relating to data interpretation, technical difficulties, genotype discrimination, and restricted amount of polymorphism (Ohresser et al. 1997). In order to analyze intraspecific phenomena related to both natural genetic substructuring and anthropogenic changes, it is useful to develop highly informative markers, such as microsatellites.

Genetic Variation Underlying Protein Expression in Eggs of the Marine Mussel Mytilus edulis

Study of the genetic basis of gene expression variation is central to attempts to understand the causes of evolutionary change. Although there are many transcriptomics studies estimating genetic variance and heritability in model organisms such as humans there is a lack of equivalent proteomics studies. In the present study, the heritability underlying egg protein expression was estimated in the marine mussel Mytilus. We believe this to be the first such measurement of genetic variation for gene expression in eggs of any organism. The study of eggs is important in evolutionary theory and life history analysis because maternal effects might have profound effects on the rate of evolution of offspring traits. Evidence is presented that the egg proteome varies significantly between individual females and that heritability of protein expression in mussel eggs is moderate to high suggesting abundant genetic variation on which natural selection might act. The study of the mussel egg proteome is also important because of the unusual system of mitochondrial DNA inheritance in mussels whereby different mitochondrial genomes are transmitted independently through female and male lineages (doubly uniparental inheritance). It is likely that the mechanism underlying this system involves the interaction of specific egg factors with sperm mitochondria following fertilization, and its elucidation might be advanced by study of the proteome in females having different progeny sex ratios. Putative identifications are presented here for egg proteins using MS/MS in Mytilus lines differing in sex ratio. Ontology terms relating to stress response and protein folding occur more frequently for proteins showing large expression differences between the lines. The distribution of ontology terms in mussel eggs was compared with those for previous mussel proteomics studies (using other tissues) and with mammal eggs. Significant differences were observed between mussel eggs and mussel tissues but not between the two types of eggs.

Proteomic Analysis of Eggs from Mytilus edulis Females Differing in Mitochondrial DNA Transmission Mode

Many bivalves have an unusual mechanism of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) in which distinctly different genomes are inherited through the female (F genome) and male (M genome) lineages. In fertilized eggs that will develop into male embryos, the sperm mitochondria remain in an aggregation, which is believed to be delivered to the primordial germ cells and passed to the next generation through the sperm. In fertilized eggs that will develop into female embryos, the sperm mitochondria are dispersed throughout the developing embryo and make little if any contribution to the next generation. The frequency of embryos with the aggregated or dispersed mitochondrial type varies among females. Previous models of DUI have predicted that maternal nuclear factors cause molecular differences among unfertilized eggs from females producing embryos with predominantly dispersed or aggregated mitochondria. We test this hypothesis using females of each of the two types from a natural population. We have found small, yet detectable, differences of the predicted type at the proteome level. We also provide evidence that eggs of females giving the dispersed pattern have consistently lower expression for different proteasome subunits than eggs of females giving the aggregated pattern. These results, combined with those of an earlier study in which we used hatchery lines of Mytilus, and with a transcriptomic study in a clam that has the DUI system of mtDNA transmission, reinforce the hypothesis that the ubiquitin-proteasome system plays a key role in the mechanism of DUI and sex determination in bivalves. We also report that eggs of females giving the dispersed pattern have higher expression for arginine kinase and enolase, enzymes involved in energy production, whereas ferritin, which is involved in iron homeostasis, has lower expression. We discuss these results in the context of genetic models for DUI and suggest experimental methods for further understanding the role of these proteins in DUI.

Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis

Proteomic analysis on sperm has been restricted to only a few model organisms. We present here a 2DE PAGE proteome map of sperm cells from a nonmodel organism, the marine mussel Mytilus edulis, a free‐spawning marine invertebrate with external fertilization. Ninety‐six protein spots showing high expression were selected and of these 77 were successfully identified by nESI–MS analysis. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results and proteomics approach used are discussed in relation to their potential for advancing understanding of the unusual system of mtDNA inheritance described in Mytilus spp., and for the testing of evolutionary hypotheses pertaining to the role of fertilization in the speciation process.

Subtle tissue and sex‐dependent proteome variation in mussel (Mytilus galloprovincialis) populations of the Galician coast (NW Spain) raised in a common environment

The mussel Mytilus galloprovincialis is one of the most important marine resources for aquaculture in Europe, and Galicia (NW Spain) is the EUs leading region for production. Variation in environmental and ecological factors exists in Northern and Southern estuaries of this region, and natural selection could have modulated genetic variation among populations with adaptation to local conditions as the driving force. Results from a previous genetic study using neutral markers suggested subtle genetic differentiation between mussel populations from both estuarine areas. In this new study, mussel samples from Northern and Southern estuaries were brought into a common environment to test for proteome differences due to genetic and permanent non‐genetic effects in populations from both estuarine areas, using both foot and mantle border tissues. Because the sex of the mussels was determined through histological tests, sex‐specific effects were also examined. Evidence of subtle differences in the foot proteome, dependent on mussel sex, were detected between populations from both estuaries. These differences were more marked for female samples. No evidence of proteome differences was found for the factors estuaries and sex in mantle border tissue. Candidate proteins with a potential role in local adaptation were identified and point to molecular functions that might be involved in responses to different stressors.

Structure-Related Differences between Cytochrome Oxidase I Proteins in a Stable Heteroplasmic Mitochondrial System

Abstract Many bivalve species have two types of mitochondrial DNA passed independently through the female line (F genome) and male line (M genome). Here we study the cytochrome oxidase I protein in such bivalve species and provide evidence for differences between the F and M proteins in amino acid property values, particularly relating to hydrophobicity and helicity. The magnitude of these differences varies between different regions of the protein and the change from the ancestor is most marked in the M protein. The observed changes occur in parallel and in the same direction in the different species studied. Two possible causes are considered, first relaxation of purifying selection with drift and second positive selection. These may operate in different ways in different regions of the protein. Many different amino acid substitutions contribute in a small way to the observed variation, but substitutions involving alanine and serine have a quantitatively large effect. Some of these substitutions are potential targets for phosphorylation and some are close to residues of functional importance in the catalytic mechanism. We propose that the observed changes in the F and M proteins might contribute to functional differences between them relating to ATP production and mitochondrial membrane potential with implications for sperm function.

Limited proteomic response in the marine snail Melarhaphe neritoides after long-term emersion

Abstract Rocky intertidal organisms are commonly exposed to environmental gradients, promoting adaptations to these conditions. Emersion time varies along the intertidal range and in the supralittoral zone is frequently larger than a single tidal cycle, even lasting for weeks. The planktonic-dispersing gastropod Melarhaphe neritoides is a common species of the high shore, adapted to reduce water loss in order to survive during long-term emersion. In this study, we investigated the molecular response, at the proteome level, of M. neritoides collected in high-shore tide pools to a series of emersion periods, from 8 to 24 days, in laboratory conditions. We compared this response to individuals maintained submerged during this period, because this was their original habitat. We also included a reversion treatment in the study, in which emersed individuals were returned to the submerged conditions. Although we detected an increase in overall protein concentration with longer emersion periods, contrary to general expectation, the two dimensional electrophoresis (2DE)-based proteomic analysis did not show significant differences between the treatments at the level of individual protein spots, even after an emersion period of 24 days. Our results suggest that the metabolism remains unaltered independent of the treatment carried out or the changes are very subtle and therefore difficult to detect with our experimental design. We conclude that M. neritoides could be equally adapted to emersion and submersion without drastic physiological changes.