All ETDs from UAB

Advisory Committee Chair

R Douglas Watson

Advisory Committee Members

Asim K Bej

Robert Roer

Louis R D'Abramo

Thane Wibbels

Document Type

Dissertation

Date of Award

2018

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

In the blue crab (Callinectes sapidus), molting, and subsequent incremental growth, is triggered by the release of molting hormones, or ecdysteroids, from paired endocrine glands termed Y-organs. The method that triggers the increase in ecdysteroid release before molting is not fully resolved, but previous evidence suggests a calcium (Ca2+) signal may be involved. The goal of this research is to better understand the role Ca2+ signaling plays in blue crab molt hormone production. The first part of my dissertation focused on the Ca2+ regulatory protein sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which removes Ca2+ to the ER after a Ca2+ influx. Using PCR-based cloning, we sequenced a putative C. sapidus SERCA cDNA from Y-organs. This produced a 3057 bp open reading frame which encoded a 1017 residue protein (Cas-SERCA). Using eyestalk ablation to remove the suppressive effects of the regulatory neuropeptide molt-inhibiting hormone (MIH), followed by quantitative real-time PCR (QPCR), we observed a temporary increase in Cas-SERCA mRNA abundance. We then applied the same QPCR assay to observe Cas-SERCA mRNA abundance in the Y-organs during a natural molt cycle. Cas-SERCA cDNA increased during pre-molt, peaked prior to molting, then gradually decreased after molting in a manner consistent with changes in the levels of intracellular Ca2+. Additionally, quantification by radioimmunoassay of hemolyphatic ecdysteroids in donor crabs showed ecdysteroid levels increased during pre-molt according to the same pattern as the increases in Ca2+ levels and Cas-SERCA mRNA abundance in Y-organs. The combined data are consistent with the hypothesis that Cas-SERCA is involved in responding to increased Ca2+ levels, but not a driving factor for the Ca2+ signal. To better understand what signaling pathways are involved in increased ecdysteroid production and release in the Y-organs, we designed an RNAseq experiment, producing a de novo transcriptome from blue crab Y-organs, and compared differentially expressed genes in pre-molt versus intermolt crabs. This experiment provided insight into possible mechanisms of ecdysteroid secretion and showed multiple signaling pathways in Y-organs that utilize Ca2+.

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