High proteome variation between ecotypes of Littorina saxatilis cannot be explained by tissue heterogeneity or a common-garden × ecotype effect

In small organisms where gene expression analyses are accomplished on whole specimens rather than individual tissues, the differences observed in gene expression levels between groups of samples are assumed to be caused by regulatory differences in gene expression within tissues. An alternative explanation is the lack of homogeneity distribution of the different tissues across groups of samples. In the case of the marine snail Littorina saxatilis, previous proteomic studies suggested a high differentiation in protein expression between the two (RB and SU) sympatric ecotypes existing on exposed rocky shores of the Galician coast (NW of Spain). As these ecotypes are known to differ in the proportion of muscular section (foot) contributing to the whole body, it remains to be checked which of the above explanations better explains this high proteome differentiation. Results from this new study suggest that tissue heterogeneity cannot explain the high proteomic differences observed between ecotypes of L. saxatilis. On the other hand, different estimates of proteome differentiation between ecotypes were obtained depending on the number of replicates used in each study (ranging from 7 to 30%). A reanalysis of previous published data shows a clear positive relationship between the degree of proteome differentiation observed and the number of biological replicates used in the analysis. This stresses the importance of investigating the effect of sample size on gene expression analyses. Finally, the results from the present study also discard the idea that observed proteome differences between ecotypes under similar laboratory conditions might be due to an interaction effect between common garden and the two different ecotypes. General conclusions drawn from the present study could be useful for setting up future gene expression studies in other species.