Calbindin-D32k in the Echinoderm Nervous System Figure 1. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the nervous system of Holothuria glaberrima (Holothuroidea). Transverse sections through the radial nerve cords showing immunoreactivity to anti-calbindin 1 (A), anti-parvalbumin (B), and anti-calbindin 2 (C) in the ectoneural and hyponeural components of the radial nerve. Double-labeling of anti-calbindin 1 (D) and RN1 (G), anti- parvalbumin (E) and RN1 (H), and anti-calbindin 2 (F) and RN1 (I) in cells (arrows) and fibers of the connective tissue plexus of the tube feet. Co- labeling is observed with the anti-CBPs markers and RN1 in the majority of the fibers, but not in all (arrowheads). ern, ectoneural component of the radial nerve cord; hrn, hyponeural component of the radial nerve cord; *, ectoneural component cell bodies; **, hyponeural component cell bodies. doi:10.1371/journal.pone.0032689.g001 GeneBank database (Echinodermata- TaxID: 7586), SpBase (the sea urchin Strongylocentrotus purpuratus genome annotation database), the S. purpuratus genome project [36], and the sea cucumber H. glaberrima expressed sequences tags EST database from our laboratory [37]. According to these databases only a putative calbindin 2 protein was found in the genome of S. purpuratus. Interestingly, no echinoderm sequences annotated for calbindin 1 or parvalbumin were found. However, we did find a secretagogin, which belongs to the calbindin subfamily of CBPs. To further characterize the most likely antigens being recognized by these Figure 2. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the peripheral nerve and circular muscle of Holothuria glaberrima (Holothuroidea). Longitudinal sections through the body wall showing colabeling of anti-calbindin 1 (A) and RN1 (D), anti- parvalbumin (B) and RN1 (E), and anti-calbindin 2 (C) and RN1 (F). Most of the immunoreactivity was observed in the peripheral nerves (arrowheads), while only a minor supopulations of fibers were co-labeled by the anti-CBP and RN1 (arrows). doi:10.1371/journal.pone.0032689.g002 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32689