All ETDs from UAB

Advisory Committee Chair

R Douglas Watson

Advisory Committee Members

Asim K Bej

Richard M Dillaman

Chi-Ying Lee

Thane Wibbels

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

In crustaceans, cellular events that lead to molting are triggered by steroid molting hormones, ecdysteroids. The synthesis of ecdysteroids is negatively regulated by molt-inhibiting hormone (MIH) mediated by one or more cyclic nucleotide second messengers. Existing data indicate ecdysteroidogenesis is positively regulated by Ca2+ through the activation of phosphodiesterases (PDEs). The studies presented in this dissertation composed the first report of intracellular Ca2+ measurement in Y-organ cells from any crustacean species. Studies demonstrated that elevated cytosolic Ca2+ levels in Y-organ cells were associated with increased hemolymphatic ecdysteroid titer. Measurement of Ca2+ levels in Y-organ cells in a natural molting cycle revealed a molting stage-specific change: Ca2+ level was low in intermolt (C), increased in premolt (D), and declined to an insignificant level in postmolt (A) stage. Molting-stage specific changes of hemolymphatic ecdysteroid titers showed a similar pattern. These combined data were consistent with the hypothesis that ecdysteroidogenesis was stimulated by an increase in intracellular Ca2+. In eukaryotic cells intracellular Ca2+ is tightly regulated by the interaction of diverse Ca2+ regulatory proteins. Among them, plasma membrane Ca2+ ATPase (PMCA), which pumps Ca2+ outside the cell, certainly plays a crucial role. In the parallel study, a putative PMCA gene (Cas-PMCA) was cloned from Callinectes sapidus Y-organs. Its deduced amino acid sequence represented all signature domains of an authentic PMCA including phosphorylation, and ATP, and calmodulin (CaM) binding sites. The transcript of Cas-PMCA was extensively detected in both neural and nonneural tissues, suggesting an essential role in various cell types. Transcript abundance of Cas-PMCA was measured in Y-organs, nonmineralization and mineralization hypodermal tissues in a natural molting cycle. Results showed that expression pattern of Cas-PMCA in Y-organs and nonmineralizing hypodermis were similar: it was low in intermolt stage, significantly increased in premolt stage and then back to resting level in postmolt stage. However, it showed an opposite expression pattern in mineralizing hypodermis. It was suggested that the elevated Cas-PMCA transcript abundance in Y-organs and nonmineralizing hypodermis might be a systemic response to the increased calcium influx when the exoskeleton is undergoing decalcification.

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