Western blots Immunoblots of nerve cord homogenates, from the sea cucumber H. glaberrima and the sea urchin L. variegatus, revealed a single immunoreactive band of approximately 32-kDa to the polyclonal anti-calbindin 1. Positive controls of mouse kidney homogenates showed a major immunoreactive band of 28-kDa (Fig. 7a). Interestingly, the immunoreactive band in a western blot of nerve cord homogenates performed with the polyclonal anti- parvalbumin, was similar to the one observed in the anti-calbindin 1 western blot; while in positive controls, using mouse skeletal muscle homogenates, a main band of approximately 12-kDa was observed (Fig. 7b). No immunoreactive band in nerve cord homogenates was observed in western blots done with the polyclonal anti-calbindin 2. Nonetheless, this antibody did not react either with homogenates of mouse kidney (the tissue from which the antigen was obtained) indicating that the antibody does not work in western blot. No bands were observed in the negative control (EFHD2 synthetic protein) demonstrating the specificity of the antigens recognized by these antibodies. The previous results prompted us to search for the H. glaberrima ortholog of the predicted calbindin-D32k identified in echinoderms (XM_001177659.1). Using primers designed against a well- conserved region between S. purpuratus calbindin-D32k (XM_0011 77659.1), Drosophila melanogaster calbindin-D32k (NM_057490.3) and Rattus norvegicus calbindin 2 (NM_053988.1), we amplified a fragment of the H. glaberrima ortholog. The amplicon was a 376 bp region of the H. glaberrima calbindin-D32k homolog, (Hg_Calb32) containing 3 out of the 6 EF-hand domains identified in S. purpuratus Figure 7. Western blot of holothurian and echinoid radial nerve cords preparations using anti-calbindin 1 and anti- parvalbumin. (A) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in H. glaberrima and L. variegatus radial nerve cords homogenates. A 28 kDa immunoreactive band corresponding to calbindin 1 was observed in the rat kidney positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain-containing protein negative control. (B) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in H. glaberrima and L. variegatus radial nerve cords homogenates. A 12 kDa major immunoreactive band corresponding to calbindin 1 was observed in the rat skeletal muscle positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain- containing protein negative control. doi:10.1371/journal.pone.0032689.g007 Calbindin-D32k in the Echinoderm Nervous System calbindin-D32k (Fig. 8a). A homology search for the peptide sequence, resulted in the top hits being calbindin-D32k sequences in invertebrates, followed by calbindin 1 and calbindin 2 sequences in vertebrates. The top two hits were the two isoforms of calbindin- D32k of S. purpuratus with e values of 2e-68 (isoform 1) and 9e-64 (isoform 2), confirming that we had successfully identified a fragment of H. glaberrima’s calbindin-D32k. Even though the cloned fragment was almost identical to isoform 1 of S. purpuratus, it still holds some interesting differences. The main difference between isoform 1 and isoform 2 from the sea urchin is a 15 amino acid long sequence from 195 to 210, which is the region we selected for the reverse degenerate primer. As can be seen from the alignment of the isoforms with Hg_Calb32, our fragment corresponded to the equivalent of isoform 1 as it has the beginning of the 15aa region. Interestingly, this is a very variable region amongst the different calbindin sequences, and only isoform 1 seems to have this 15 amino acid motif. There are 6 variable residues between sea cucumber and sea urchin sequence. Of these, the addition of an amino acid in Hg_Calb32 at position 93 or 94 absent in the sea urchin sequences stands out, as it is not present in any of the other sequences. Hg_Calb32 was cloned and expressed in an heterologous system. As our construct contained a GST tag, we employed Affinity Chromatography for the purification of the Hg_Calb32, which was then analyzed through SDS-PAGE. Coomassie staining showed enrichment of a ,36 kDa band, which is near the expected weight of our fragment tagged with GST. A western blot of the expressed peptide tagged with GST was immunopositive to both anti-calbindin 1 and anti-parvalbumin, in the expected band size of 36 kDa (Fig. 8b), as well as when proved with an anti-GST antibody. No immunoreactive band was observed in the negative control, which consisted of GST tagged TCTP when incubated with anti-calbindin 1 or anti-parvalbumin (Fig. 8b), which was immunopositive when proved with the anti-GST antibody. Discussion In this study we report the distribution of an EF-hand domain containing CBP of the calbindin subfamily in the nervous system of the echinoderms. This is the first report that describes the distribution of these proteins, which have been well-studied in the vertebrate nervous system, in the echinoderm nervous system. Accordingly, calbindin-D32k is specifically localized within the nervous system of the echinoderm H. glaberrima, extending the usefulness of this neural marker to invertebrates, in particular to the Echinodermata. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity is restricted to the nervous system The immunoreactivity of these CBP markers is specific to echinoderm neural components. The evidence for this is the following: First, in other organisms in which these have been studied (mostly mammals), these markers specifically label neural structures [9,10,16]. Second, in the holothurian studied here, immunoreactivity to these markers is observed in cells and fibers of the radial nerve cords, podial nerves and peripheral nerves, which are the classically described neural structures in the echinoderms [27,29,30,38–40]. Finally, double-labeling experiments with known neural markers (RN1 and anti-b-tubulin), and serial sections done with markers made against the neuropeptides GFSKLYamide and Galanin show co-labeling of the same fibers and cells by the CBP markers and the neural markers. Similarly, the localization of some of the fibers and cells labeled by anti- GFSKLYamide and anti-Galanin is similar to that of the three CBP markers in serial sections. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e32689