antigen. Anti-calbindin 1 immunoreactivity was more intense than that of anti-parvalbumin or anti-calbindin 2, which is most likely the result of a higher affinity of the anti-calbindin 1 antibody for the antigen being recognized. Thus when taking these observa- tions into account, in addition to the fact that no parvalbumin orthologue was identified in the S. purpuratus genome, the most plausible explanation for the parvalbumin immunoreactivity in the echinoderms is that this antibody is recognizing a conserved sequence of the EF-hand domain containing CBP in one of the calbindin subfamily members. We tested this hypothesis, and showed that both markers are recognizing a very similar, if not the same, antigen in radial nerve western blot. Furthermore, the H. glaberrima calbindin-D32k fragment western blots showed that the antigen in question is the orthologue of calbindin 2 in echinoderms, calbindin-D32k. These last sets of experiments show that indeed calbindin-D32k is present in the echinoderms and that it is the antigen being recognized by the anti-calbindin 1 and anti- parvalbumin markers used in our study. This last observation possibly holds true for the anti-calbindin 2 marker as well, though we only able to gather indirect evidence supporting this. Calbindin-D32k is present in Echinodermata We have provided evidence of the presence of calbindin-D32k within the echinoderms, and this is the first report that establishes its presence in Echinodermata. Calbindin-D32k was also previ- ously identified in Drosophila [13], although due to the lack of the full genome of the organism at the time, no evolutionary relation could be made with the members of the calbindin subfamily of EF- hand domain containing CBPs. The calbindin-D32k identified in Drosophila was named for its apparent molecular weight of 32 kDa in SDS-PAGE. Interestingly, calbindin-D32k and secretagogin are the only members of the calbindin subfamily present in invertebrates. Thus, our results also support the evolutionary conservation of the calbindin subfamily, which seems to have evolved from two primitive EF-hand domain-containing CBP. One of them gave rise to calbindin-D32k in invertebrates and to calbindin 1 and calbindin 2 in vertebrates, while the other gave References 1. Andressen C, Blumcke I, Celio MR (1993) Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271: 181–208. 2. Baimbridge KG, Celio MR, Rogers JH (1992) Calcium-binding proteins in the nervous system. Trends Neurosci 15: 303–308. 3. Bennett-Clarke CA, Chiaia NL, Jacquin MF, Rhoades RW (1992) Parvalbumin and calbindin immunocytochemistry reveal functionally distinct cell groups and vibrissa-related patterns in the trigeminal brainstem complex of the adult rat. J Comp Neurol 320: 323–338. 4. 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This hypothesis is also supported by findings from genomic sequencing of model organisms such as D. melanogaster, S. purpuratus or S. kowalevskii, in which only one ortholog of calbindin-D32k and one of secretagogin have been identified [17]; as well as previous works suggesting that calbindin 1 arose during the radiation of vertebrates and is a vertebrate-specific gene [13,16,19]. Nonethe- less, there is still a need for a major pool of sequences from a wider variety of organisms to reach a better-informed conclusion, of when specifically did calbindin 1 arose. Thus we propose that the vertebrate calbindin 1 and calbindin 2 are co-orthologous of the calbindin-D32k of Drosophila and the calbindin-D32k of S. purpuratus and H. glaberrima. In conclusion, our study contributes to a better understanding of the evolution of the calbindin subgroup of CBPs through the species and their role in the nervous system. We have identified a calbindin 2 ortholog in two echinoderms classes, Holothuroidea and Echinoidea, which is associated with the nervous system, more specifically with the connective tissue plexi. This protein corresponds to the calbindin-D32k previously identified in Drosophila, which is the ortholog of calbindin 2. Additionally, given the similarities found in sequence and structure, we show that the calbindin 1, parvalbumin, and calbindin 2 commercial antibodies are labeling the invertebrate ortholog of calbindin 2, calbindin-D32k in the echinoderm nervous system. Acknowledgments We would like to thank Natalia Almodovar and Griselle Valent´ın-Tirado for their help with the immunohistochemistry and western blot experiments, and Pablo Ortiz-Pineda for his help with the Bioinformatics and critical reading of the manuscript. Author Contributions Conceived and designed the experiments: CADB MILP CIG JEGA. Performed the experiments: CADB MILP EMGR. Analyzed the data: CADB MILP JEGA. 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(1997) Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B. Protein Sci 6: 2397–2408. PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e32689