Meeting Abstract

8-3  Thursday, Jan. 5 09:00 – 09:15  Cloning of a cDNA Encoding a Sarco/endoplasmic Reticulum Ca²⁺ ATPase (SERCA) from Y-organs of the Blue Crab (Callinectes sapidus) and Spatial and Temporal Patterns of SERCA Expression ROEGNER, ME*; CHEN, HY; WATSON, RD; University of Alabama at Birmingham; University of Alabama at Birmingham mzappe@uab.edu

Stage-specific increases in intracellular free Ca²⁺ have been shown to stimulate ecdysteroid production in the molting glands (Y-organs) of crustaceans. Intracellular Ca²⁺ levels are regulated by proteins intrinsic to the plasma membrane and membranes of organelles. These include Ca²⁺ pumps, e.g., plasma membrane calcium ATPases (PMCAs) and sarco/endoplasmic reticulum calcium ATPases (SERCAs). In order to better understand the role of intracellular calcium signaling in the regulation of ecdysteroidogenesis, we used a PCR based cloning strategy (RT-PCR followed by 3′- and 5′-RACE) to clone a full length cDNA encoding a putative SERCA protein from the Y-organs of the blue crab (Callinectes sapidus). The cDNA includes an optimal translational initiation sequence, a start codon, and a 2488-bp open reading frame that encodes an 829-residue protein with 91 percent identity to comparable crustacean SERCA sequences. Phylogenetic analysis shows the blue crab SERCA protein to cluster with other arthropod SERCAs. An assessment of spatial distribution showed the SERCA transcript to be widely distributed across tissues of the crab. Furthermore, SERCA transcript levels were analyzed in Y-organs using quantitative PCR. Transcript abundance was assessed after Y-organs were activated by eyestalk ablation, and throughout a natural molting cycle. Stage specific changes in SERCA cDNA levels provide insight into the role of Ca²⁺ signaling and intracellular Ca²⁺ regulatory proteins in endocrine regulation of crustacean molting.