Calcium signaling and regulation of ecdysteroidogenesis in crustacean Y-organs.

Crustacean Y-organs secrete ecdysteroid molting hormones. Ecdysteroids are released in increased amount during premolt, circulate in hemolymph, and stimulate the events in target cells that lead to molting. During much of the molting cycle, ecdysteroid production is suppressed by molt-inhibiting hormone (MIH), a peptide neurohormone produced in the eyestalks. The suppressive effect of MIH is mediated by a cyclic nucleotide second messenger. A decrease in circulating MIH is associated with an increase in the hemolymphatic ecdysteroid titer during pre-molt. Nevertheless, it has long been hypothesized that a positive regulatory signal or stimulus is also involved in promoting ecdysteroidogenensis during premolt. Data reviewed here are consistent with the hypothesis that an intracellular Ca2+ signal provides that stimulus. Pharmacological agents that increase intracellular Ca2+ in Y-organs promote ecdysteroidogenesis, while agents that lower intracellular Ca2+ or disrupt Ca2+ signaling suppress ecdysteroidogenesis. Further, an increase in the hemolymphatic ecdysteroid titer after eyestalk ablation or during natural premolt is associated with an increase in intracellular free Ca2+ in Y-organ cells. Several lines of evidence suggest elevated intracellular calcium is linked to enhanced ecdysteroidogenesis through activation of Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, thereby lowering intracellular cyclic nucleotide second messenger levels and promoting ecdysteroidogenesis. Results of transcriptomic studies show genes involved in Ca2+ signaling are well represented in Y-organs. Several recent studies have focused on Ca2+ transport proteins in Y-organs. Complementary DNAs encoding a plasma membrane Ca2+ ATPase (PMCA) and a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) have been cloned from crab Y-organs. The relative abundance of PMCA and SERCA transcripts in Y-organs is elevated during premolt, a time when Ca2+ levels in Y-organs are likewise elevated. The results are consistent with the notion that these transport proteins act to maintain the Ca2+ gradient across the cell membrane and re-set the cell for future Ca2+ signals.

Effect of a chemical dispersant (Corexit 9500A) on the structure and ion transport function of blue crab (Callinectes sapidus) gills.

Chemical dispersants are commercially available mixtures of surfactants and solvents that have become important tools in the remediation of spilled oil. Given the importance of oil to the world economy, the recurring nature of spills, and the prevalence of dispersant use in remediation, there is a critical need to understand potential toxic impacts of dispersants on invertebrate and vertebrate animals. Blue crabs (Callinectes sapidus) play ecologically important roles in the environments they inhabit and support economically important fisheries along the Atlantic Coast and in the Gulf of Mexico. In studies reported here, we assessed the impact of a chemical dispersant, Corexit 9500A, on the structure and ion transport function of blue crab gills. Exposure of blue crabs to Corexit 9500A for 24 h (0-300 ppm in artificial seawater under static conditions) revealed a 24-h lethal concentration 50 (LC50) estimate of 210 ppm. A histological analysis of gills from crabs exposed for 24 h to a sub-lethal concentration of Corexit 9500A (125 ppm) revealed evidence of loss or disruption of cuticle, and an increase in stained amorphous material in the hemolymph spaces of gill lamellae. Quantitative image analysis of stained gill sections revealed the area/length ratio of gill lamellae in crabs exposed to Corexit 9500A (24 h, 125 ppm), was greater than that in gill lamellae from control crabs; the results are consistent with the presence of edematous swelling in gill lamellae from dispersant-exposed crabs. Quantitative PCR was used to measure the relative abundance of transcripts encoding three ion transport proteins (Na+/K+ ATPase, plasma membrane Ca2+ ATPase (PMCA), and sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase (SERCA)) in gills from Corexit-exposed and control crabs. In general, the abundance of transcripts encoding each ion transport protein was lower in gills from dispersant-exposed crabs than in gills from control crabs. The combined results are consistent with the hypothesis that 24-h exposure of blue crabs to a sublethal concentration of Corexit 9500A impacts both the structure and ion transport function of gills.

De novo transcriptome assembly and functional annotation for Y-organs of the blue crab (Callinectes sapidus), and analysis of differentially expressed genes during pre-molt.

Blue crabs (Callinectes sapidus) undergo incremental growth involving the shedding (molting) of the old exoskeleton, and subsequent expansion and re-calcification of the newly synthesized one. The cellular events that lead to molting are triggered by steroid hormones termed ecdysteroids released from Y-organs, paired endocrine glands located in the anterior cephalothorax. The regulatory pathways leading to increased synthesis and release of ecdysteroids are not fully understood, and no transcriptome has yet been published for blue crab Y-organs. Here we report de novo transcriptome assembly and annotation for adult blue crab Y-organs, and differential gene expression (DGE) analysis between Y-organs of intermolt and premolt crabs. After trimming and quality assessment, a total of 91,819,458 reads from four cDNA libraries were assembled using Trinity to form the reference transcriptome. Trinity produced a total of 171,530 contigs coding for 150,388 predicted genes with an average contig length of 613 and an N50 of 940. Of these, TransDecoder predicted 31,661 open reading frames (ORFs), and 10,210 produced non-redundant blastx results through Trinotate annotation. Genes involved in multiple cell signaling pathways, including Ca2+ signaling, cGMP signaling, cAMP signaling, and mTOR signaling were present in the annotated reference transcriptome. DGE analysis showed in premolt Y-organs up-regulated genes involved in energy production, cholesterol metabolism, and exocytosis. The results provide insights into the transcriptome of blue crab Y-organs during a natural (rather than experimentally induced) molting cycle, and constitute a step forward in understanding the cellular mechanisms that underlie stage-specific changes in the synthesis and secretion of ecdysteroids by Y-organs.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transcript abundance in Y-organs and ecdysteroid titer in hemolymph during a molting cycle of the Blue Crab, Callinectes sapidus.

Crustacean growth is characterized by molting, whereby the old exoskeleton is shed and replaced by a new and larger version. The cellular events that lead to molting are driven by steroid hormones (ecdysteroids) secreted by paired endocrine glands (Y-organs). Between molts, ecdysteroid production is suppressed by a polypeptide molt-inhibiting hormone (MIH) released from neurosecretory cells in the eyestalks. Although a decrease in the MIH titer precedes the upsurge in ecdysteroidogenesis, it is hypothesized that a positive regulatory signal is also required for full activation of Y-organs. Existing data point to an intracellular Ca2+ signal. Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, achieved through membrane transport proteins. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the ER. We have recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative Cas-SERCA protein. In studies reported here, quantitative PCR (QPCR) was used to quantify Cas-SERCA transcript abundance in Y-organs during a molting cycle, and radioimmunoassay was used to quantify ecdysteroids in hemolymph. The abundance of the Cas-SERCA transcript in Y-organs increased gradually during pre-molt. Similarly, the level of ecdysteroids in hemolymph increased during pre-molt. The results are consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs. Cas-SERCA transcript abundance also changed in several non-ecdysteroidogenic tissues during a molting cycle. The pattern of change differed among tissues suggesting a functional role for SERCA in each.

Molecular cloning and characterization of a sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from Y-organs of the blue crab (Callinectes sapidus).

Existing data indicate that a Ca2+ signal stimulates ecdysteroid hormone production by crustacean molting glands (Y-organs). Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, with intracellular free Ca2+ maintained at a low basal level (typically sub-micromolar). This is achieved through the action of proteins intrinsic to the plasma membrane and the membranes of organelles. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the endoplasmic reticulum. As a step toward understanding Ca2+-mediated regulation of ecdysteroidogenesis, we have begun investigating Ca2+ transport proteins in Y-organs. In studies reported here, we used a PCR-based strategy to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative SERCA protein. The cloned Cas-SERCA cDNA (3806 bp) includes a 3057-bp open reading frame that encodes a 1019-residue protein (Cas-SERCA). The conceptually translated protein has a predicted molecular mass of 111.42 × 103 and contains all signature domains of an authentic SERCA, including ten transmembrane domains and a phosphorylation site at aspartate 351. A homology model of Cas-SERCA closely resembles models of related SERCA proteins. Phylogenetic analysis shows Cas-SERCA clusters with SERCA proteins from other arthropods. An assessment of tissue distribution indicates the Cas-SERCA transcript is widely distributed across tissues. Studies using quantitative PCR showed Cas-SERCA transcript abundance increased significantly in Y-organs activated by eyestalk ablation, a pattern consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs.