Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea.

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Molecular modification of Penaeus monodon sperm in female thelycum and its consequent responses.

Using Penaeus monodon as the model, we demonstrated the molecular changes and the mechanism of thelycal-dependent sperm modification resulting in an enhanced acrosome reaction (AR) response. Attention was paid to the modification of the sperm plasma membrane which was mediated through an adsorption or removal of sperm peripheral and integral membrane proteins as indicated by the different profiles of these proteins in spermatophore (S) and thelycal (T) sperm. In vitro adsorption of Alexa-488 conjugated T proteins onto the entire S-sperm surface confirmed protein transfer in a time-dependent manner. Specific anchoring of 83 and 140 kDa proteins to sperm peripheral proteins as well as 53/55 and 60 kDa proteins to sperm lipids was demonstrated. Apart from membrane modification, a substantial increase in protein tyrosine phosphorylation was shown to be closely associated with T-dependent sperm modification event. The physiological significance of this sperm modification in enhancing sperm AR response, which required at least 3 days of T residence in order for the sperm to gain a complete AR response, was also elucidated.

Effects of controlled pH on organic and inorganic composition in haemolymph, epidermal tissue and cuticle of mud crab Scylla serrata.

Analysis of organic and inorganic compounds in plasma, epidermal tissue and cuticle were accomplished in the intermolt (C3 stage) of crab Scylla serrata incubated in different pH media. Significant changes with similar trends for protein, carbohydrates, glycosaminoglycans (GAG), sulphur, calcium, magnesium, potassium, phosphorus and copper in the plasma suggested higher dissolution in an acidic medium while the deposition increased in alkaline medium. Similar decreases in protein, carbohydrate and GAG in the epidermal compartment were observed from pH 4 to pH 12. However, significantly higher contents of sodium, chloride, potassium, phosphorus, magnesium, sulphur and copper were measured at pH 7.5 with a symmetrical decrease profile in both acidic and alkaline media, resulting from synergistic effects in the osmotic regulation. Clear changes in calcium concentrations were observed with a sharp increase from lower contents at pH 7.5 to higher at pH 12. In the cuticle, the acidic condition induced a significant dissolution of HCl-protein, GAG, calcium and magnesium contents. On the other hand, the alkaline condition induced a significant decrease in carbohydrate, calcium, chloride, sulphur and potassium. A reduction trend is seen for NaOH and H(2)O-protein contents in the cuticle. These observations suggest that GAG and HCl-protein might constitute the most soluble fraction with high affinity for calcium binding and easily removed in acidic conditions. Additionally, it is possible to speculate that the carbohydrates associated with the NaOH and H(2)O-proteins may form an interface between the soluble matrix fraction and the chitin framework. Sulphur groups seem to present a strong linkage role in this interface fraction, maybe only broken by a specific enzyme in extreme alkaline conditions with subsequent release of significant calcium from the shell.

Cuticle Ultrastructure Changes in the Crab Scylla serrata Over the Molt Cycle.

Morphological and chemical studies on the cuticle during the molt cycle of the crab Scylla serrata were performed in order to understand the layer formation. Cuticle ultrastructure was studied by scanning electron microscopy (SEM). Energy-dispersive, X-ray diffraction, and X-ray fluorescence analysis were used for identification of the elements and phases in the inner surface of the cuticle. In the first stage (A) of cuticle formation, a thin pellicle organized as an irregular fragmented structure is built. It is composed mainly of alpha-chitin/protein beta-keratin-like complexes where heterogeneous mineral nucleation occur. It is impregnated by ferric concretions, responsible for the brown colour of the carapace. At the beginning of the mineralization process, a spheroidal inorganic phase appears consisting of dicalcium phosphate dihydrate (DCPD) Ca/P=1.00, octacalcium phosphate (OCP) Ca/P=1.33 associated with hydromagnesite and bromapatite traces. During further cuticle development in the remaining A stage and in the beginning of the B stage, calcite and magnesian calcite are formed from the precursor calcium phosphate phase. The next development in the C stages is characterized by intense calcareous thickening consisting mainly of calcite and of magnesian calcite, which become the major mineral fraction of the cuticle. Organic-inorganic complex precipitations exhibit different aspects as spongiform, filamentary helicoidal, and concentric radial arrangements during C1, C2, and C3, respectively. During different stages of the cuticle formation in Scylla serrata, these mineral deposits may partially result from the balance among different organic contents, mainly between alpha-chitin and protein beta-keratin-like compounds. On the other hand, the calcium crystallization on apatite and calcite polymorphic structures may be influenced by variations of physico-chemical factors in the cuticle compartment. J. Exp. Zool. 293:414-426, 2002.

Organic and inorganic compound variations in haemolymph, epidermal tissue and cuticle over the molt cycle in Scylla serrata (Decapoda).

Analysis of organic and inorganic compounds in haemolymph, epidermal tissue and cuticle, allowed the correlation of their content variation during the molt cycle in Scylla serrata (Decapoda). The extrusion of carbohydrate, chitin, H(2)O- and NaOH-soluble proteins of the epidermal tissue from D2 stage on suggested an early formation of the organic fraction in the new cuticle. The additional secretion of HCl-soluble protein, glycosaminoglycans, calcium, phosphorus and sulfur in D2-D3 stages suggests mineral nucleation shortly before or at ecdysis. This is consistent with the dominant content of proteins and chitin associated with peaks of carbohydrates and GAG-sulfur groups detected in the cuticle immediately after ecdysis. Furthermore, the maximal phosphorus content at this moment suggests calcium, magnesium and manganese phosphate deposits. After an intense accumulation of calcium, chitin and sharp decrease of protein from A to B stages, a constant rate of their deposition in intermolt and dissolution in premolt occurred. The variations concerning proteins, carbohydrates, glycosaminoglycans, calcium, magnesium and sulfur in the haemolymph, before and after ecdysis, suggest a transitory phenomenon for calcium binding and releasing. Other major elements such as, sodium, potassium and chloride may function as osmotic regulators in the haemolymph and in epidermal compartments. The copper profile presented an indicator role for variations of osmolality over the molt cycle.