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.

Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

Molecular cloning of a plasma membrane Ca²âº ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.

Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs.

Stage-specific changes in calcium concentration in crustacean (Callinectes sapidus) Y-organs during a natural molting cycle, and their relation to the hemolymphatic ecdysteroid titer.

Secretion of ecdysteroid molting hormones by crustacean Y-organs is suppressed by molt-inhibiting hormone (MIH). The suppressive effect of MIH on ecdysteroidogenesis is mediated by one or more cyclic nucleotide second messengers. In addition, existing data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular Ca(++). Despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(++) in Y-organ cells has not been previously measured during a natural molting cycle for any crustacean species. In studies reported here, a fluorescent calcium indicator (Fluo-4) was used to measure Ca(++) levels in Y-organs during a molting cycle of the blue crab, Callinectes sapidus. Mean calcium fluorescence increased 5.8-fold between intermolt (C4) and stage D3 of premolt, and then dropped abruptly, reaching a level in postmolt (A) that was not significantly different from that in intermolt (P>0.05). The level of ecdysteroids in hemolymph of Y-organ donor crabs (measured by radioimmunoassay) showed an overall pattern similar to that observed for calcium fluorescence, rising from 2.9 ng/mL in intermolt to 357.1 ng/mL in D3 (P<0.05), and then dropping to 55.3 ng/mL in D4 (P<0.05). The combined results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(++).

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y-organs: relation to the hemolymphatic ecdysteroid titer.

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5 ng/mL on D0 to 49.6 ng/mL on D6 and 87.2 ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).

Molecular cloning of a putative crustacean hyperglycemic hormone (CHH) isoform from extra-eyestalk tissue of the blue crab (Callinectes sapidus), and determination of temporal and spatial patterns of CHH gene expression.

Crustacean hyperglycemic hormone (CHH) is a polypeptide neurohormone involved in regulation of multiple physiological processes. We report here the cloning from thoracic ganglia of the blue crab (Callinectes sapidus) a cDNA (CsCHH-2) encoding a putative CHH isoform (CsCHH-2). CsCHH-2 is structurally similar to a putative preproCHH (CsCHH-1) previously cloned from eyestalk ganglia of C. sapidus. The two preprohormones possess an identical signal peptide and CHH precursor related peptide, but differ in the mature CHH polypeptide. An analysis by RT-PCR of the tissue distribution of CsCHH-1 and CsCHH-2 revealed the former is restricted to eyestalk neural ganglia, while the latter is widely distributed among tissues. The type of CHH transcript present in eyestalk and thoracic ganglia did not vary as a function of the molt cycle. An assessment of transcript abundance in tissues of intermolt crabs showed the abundance of the CsCHH-1 transcript in eyestalk ganglia far exceeds the abundance of the CsCHH-2 transcript in extra-eyestalk tissue. An assessment of transcript abundance during a molt cycle showed CsCHH-1 transcript abundance in eyestalk ganglia was low during intermolt, rose during premolt, reaching a peak in D(3), then fell prior to molting, and remained low during postmolt. By contrast, CsCHH-2 transcript abundance in thoracic ganglia was low during intermolt, rose sharply during D(2), then dropped in D(3) and remained low during postmolt. The results are consistent with the hypothesis that CsCHH-1 and CsCHH-2 differ with respect to physiological function.

Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea.

Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further alpha-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower alpha-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 alpha-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.

Crustacean molt-inhibiting hormone: structure, function, and cellular mode of action.

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.

Studies of a receptor guanylyl cyclase cloned from Y-organs of the blue crab (Callinectes sapidus), and its possible functional link to ecdysteroidogenesis.

Crustacean Y-organs synthesize ecdysteroid molting hormones. Synthesis of ecdysteroids by Y-organs is negatively regulated by a polypeptide neurohormone, molt-inhibiting hormone (MIH). Our laboratory has recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA (CsGC-YO1) encoding a putative receptor guanylyl cyclase (CsGC-YO1). We hypothesize that CsGC-YO1 is an MIH receptor. In studies reported here, antipeptide antibodies (anti-CsGC-YO1) were raised against a fragment of the extracellular domain of CsGC-YO1. Western blots showed affinity purified anti-CsGC-YO1 bound to the heterologously expressed extracellular domain, and to a protein in Y-organs that corresponded in size to the theoretical molecular mass of CsGC-YO1. Immunocytochemical studies with anti-CsGC-YO1 as primary antibody, showed CsGC-YO1 immunoreactivity was restricted to the peripheral margins of cells, and was not present in cytoplasm or nuclei. The results strongly suggest that CsGC-YO1 is a membrane-associated protein. Preincubation of Y-organs with anti-CsCG-YO1 blunted MIH-induced suppression of ecdysteroidogenesis. This finding represents the first demonstration of a link between CsGC-YO1 and MIH action. A real-time PCR assay for quantifying CsCG-YO1 was developed and validated. The assay was used to determine the abundance of the CsCG-YO1 transcript in Y-organs during a molt cycle: the level of CsGC-YO1 in Y-organs was elevated during intermolt (C(4)) and lower during premolt stages D(1)-D(3). The data suggest that the biological action of CsGC-YO1 in Y-organs is likely to be most pronounced during intermolt. The combined results are consistent with the hypothesis that CsGC-YO1 is an MIH receptor.

Molecular characterization and gene expression pattern of two putative molt-inhibiting hormones from Litopenaeus vannamei.

Two cDNA sequences (Liv-MIH1 and Liv-MIH2) were cloned from the eyestalk ganglia of the white shrimp Litopenaeus vannamei. The conceptually translated peptide precursors consist of a mature peptide (77 residues for Liv-MIH1, 75 residues for Liv-MIH2), preceded by a 28-residue signal peptide. Both mature peptides share highest sequence identity with other known MIHs, and contain several conserved residues that have been proposed to be functionally critical for MIH activity. Analysis of genomic sequences reveals that both genes are organized in a 3 exon/2 intron manner, with the same sites of intron insertion. The transcripts of Liv-MIH1 and Liv-MIH2 were detected exclusively in the eyestalk, but not in other neural and non-neural tissues examined. Phylogenetic analysis indicates that Liv-MIH1 and Liv-MIH2 cluster with the type II peptides that are considered as penaeid MIH. In addition, a quantitative real-time polymerase chain reaction (PCR) assay was developed and validated for the quantification of gene expression of Liv-MIH1 and Liv-MIH2. Transcript levels for both genes remained constant through stages A - D(1') (ranges of relative expression levels are 97.9+/-2.9 to 104.5+/-8.9% for Liv-MIH1, and 85.6+/-6.7 to 104.7+/-10.8% for Liv-MIH2), and declined afterwards, reaching a lowest level during stage D(2)D(3) (40.6+/-0.4% for Liv-MIH1, and 48.5+/-3.2% for Liv-MIH2). These significant decreases in the transcript levels correspond to a significant increase in hemolymph ecdysteroid titers at stage D(2)D(3). These results clearly indicate that Liv-MIH1 and Liv-MIH2 are type II peptides of the crustacean hyperglycemic hormone family and most likely function as MIHs in the white shrimp. They are discussed with regard to the presence of multiple MIHs and possible functional divergence of type II peptides in Penaeidae, as well as endocrine regulation of crustacean molting.

Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): involvement of cyclic GMP and cyclic nucleotide phosphodiesterase.

Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Previous results indicate the biosynthetic activity of Y-organs is likely controlled not only by the level of hemolymphatic MIH, but also by the responsiveness of Y-organs to MIH. The present studies were conducted to (a) identify the second messenger that mediates MIH-induced suppression of ecdysteroidogenesis, and (b) assess the possible involvement of cyclic nucleotide phosphodiesterase (PDE) in determining the responsiveness of Y-organs to MIH. Adding 8-bromo cAMP or 8-bromo cGMP to incubation medium significantly suppressed ecdysteroid production by Y-organs of the crayfish (Procambarus clarkii). Incubating Y-organs with MIH produced a significant increase in glandular cGMP, but MIH had no effect on glandular cAMP. The composite data indicate that MIH-induced suppression of ecdysteroidogenesis in Y-organs of P. clarkii is mediated by cGMP. Subsequently, Y-organs from various stages of the molt cycle were incubated with MIH, 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of PDE), or both. Y-Organs from middle and late premolt stages were poorly responsive to MIH alone. Including IBMX in the incubation medium enhanced the responsiveness of the Y-organs to MIH at these stages. Moreover, glandular PDE activity in the Y-organs at these stages was significantly higher than other stages. The combined results suggest that molt cycle-associated changes in PDE activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of P. clarkii.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs.

A neuropeptide, molt-inhibiting hormone (MIH), negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.

Molecular cloning of a cDNA encoding a crustacean hyperglycemic hormone from eyestalk ganglia of the blue crab, Callinectes sapidus.

Crustacean hyperglycemic hormone (CHH), a polypeptide with multiple physiological effects, was first identified in the X-organ/sinus gland neurosecretory system of the eyestalks. In studies reported here, we used a PCR-based cloning strategy (RT-PCR followed by 5'- and 3'-RACE) to clone from blue crab (Callinectes sapidus) eyestalk ganglia a cDNA (CsCHH-1) encoding a putative CHH preprohormone. Sequence analysis revealed the preprohormone included all structural features previously reported for CHH preprohormones: a signal peptide, a CHH precursor-related peptide (CPRP), the CHH polypeptide, and a C-terminal basic processing site. Further, the deduced amino acid sequence of the mature polypeptide included all signature domains previously reported for CHH. The primary structure of blue crab CHH is most closely related to CHH from other brachyurans. RT-PCR revealed the CsCHH-1 transcript was present in eyestalk ganglia, but was undetectable in other tissues tested. A transcript encoding a similar CHH-like preprohormone was detected in thoracic ganglion, ventral nerve cord, and brain, but was not detected in eyestalk ganglia.

Molecular cloning of a putative receptor guanylyl cyclase from Y-organs of the blue crab, Callinectes sapidus.

Crustacean molt-inhibiting hormone (MIH), a polypeptide produced by neurosecretory cells in eyestalk ganglia, suppresses the synthesis of ecdysteroid molting hormones by paired Y-organs. Data from several sources indicate the effects of MIH are mediated, at least in part, by a cGMP second messenger. Based on these and related findings, our working hypothesis is that the MIH receptor is a receptor guanylyl cyclase (rGC). In studies reported here, we used a PCR-based cloning strategy (RT-PCR followed by 5'- and 3'-RACE) to clone from blue crab (Callinectes sapidus) Y-organs a cDNA (CsGC-YO1) encoding a putative rGC. DNA sequence analysis revealed a 3807 base pair open reading frame encoding a 56 residue signal peptide and a 1213 residue rGC. Analysis of the deduced amino acid sequence showed that CsGC-YO1 contains the signature domains characteristic of rGCs, including an extracellular ligand-binding domain, a single transmembrane domain, a kinase-like domain, a dimerization domain, and a cyclase catalytic domain. CsGC-YO1 is most closely related to an rGC from the crayfish, Procambarus claikii (PcGC-M2, 58.4% identity), and rGCs from three insect species (33.1-37.5% identity). Conserved cysteine residues are similarly distributed in the extracellular domains of CsGC-YO1, PcGC-M2, and the three insect rGCs. RT-PCR revealed the CsGC-YO1 transcript is expressed in Y-organs and several other tissues. While other interpretations of the data are possible, our working hypothesis is that the cloned cDNA encodes an MIH receptor.

Effects of 17alpha-ethynylestradiol on sexual development of male western mosquitofish (Gambusia affinis).

Juvenile male western mosquitofish (Gambusia affinis) were exposed to different concentrations of 17alpha-ethynyl estradiol (EE2) in the diet during the period of sexual maturation. A clear inhibiting influence of EE2 on sexual development was apparent. The proportion of males in each treatment group that failed to complete gonopodial development during the 150-day observation period increased significantly with EE2 concentration. There were significant nonlinear trends toward shorter gonopodia in groups exposed to higher EE2 concentrations. Vitellogenin (VTG) was detectable in the blood of all fish exposed to 1.0 or more micro/g food and the concentration increased dramatically with increasing EE2 exposure. A significant negative association was seen between EE2 concentration and spermatophore counts. This study has demonstrated deleterious effects of EE2 exposure on sexual maturation and several indirect measures of reproductive fitness. It supports the biological relevance of vitellogenin in the blood and reduced gonopodium length as biomarkers for estrogen exposure and endocrine disruption in mosquitofish.

Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis.

Crustacean Y-organs produce ecdysteroid molting hormones. Regulation of ecdysteroidogenesis appears to be complex, involving regulatory ligands (including but not limited to molt-inhibiting hormone, an eyestalk neurohormone) and the capacity of the Y-organs to respond to those ligands. Available data indicate cell signaling pathways involving cAMP, cGMP, or both may be involved in regulation of Y-organ function. Trimeric G proteins link receptor occupancy to regulation of intracellular cAMP levels. In studies reported here, we have assessed the occurrence of G proteins in blue crab (Callinectes sapidus) Y-organs, and the link of G proteins to Y-organ function. Bacterial toxin-catalyzed ADP-ribosylation revealed a PTX-sensitive (alpha i-like) protein in Y-organ membranes, but failed to reveal a CTX-sensitive (alpha s-like) protein in Y-organ membranes. Western blotting with primary antibodies raised against conserved regions of mammalian G proteins detected an alpha i-immunoreactive protein (approximately 40 kDa) and two alpha s-immunoreactive proteins (approximately 50 and approximately 57 kDa) in Y-organ membrane preparations. Incubation of Y-organ membrane fractions with cholera toxin significantly suppressed incorporation of [35S]-methionine into TCA-precipitable Y-organ proteins, but had no detectable effect on ecdysteroidogenesis in short-term (6 h) incubations. The combined results indicate that C. sapidus Y-organs possess both Gi and Gs proteins, and that alpha s is functionally linked to regulation of glandular protein synthesis.