Robert D. Roer

Distribution and Characterization of Ion Transporting and Respiratory Filaments in the Gills of Procambarus clarkii

Individual gill filaments of the freshwater crayfish Procambarus clarkii were determined to be either predominantly respiratory or transporting. Silver staining revealed that the filaments within the central bed of the gills formed silver deposits whereas filaments at the margins and the entire sixth pleurobranch formed no deposits. Designation of the silver staining gills as predominantly transporting and unstained filaments as predominantly respiratory was substantiated by ultrastructural analyses and measurements of ATPase and transepithelial potentials. Presumptive transporting filaments had an epithelium subjacent to the cuticle that was relatively thick and dominated by abundant mitochondria. Lacunae were delineated by pillar structures and served as collateral pathways for the movement of blood from the afferent to efferent blood channels, which were separated by a thin septum. Presumptive respiratory filaments had an extremely thin epithelium with few organelles, but a relatively thick septum. Present in both types of filaments were nerves and podocytes. The values for Na, K-ATPase were significantly higher in the transporting filaments than in those designated as respiratory. The measurement of transepithelial potentials showed both filaments to be cation selective with the respiratory filaments slightly more positive and the transporting filaments slightly more negative than the diffusion potential for Na.

Cuticular proteins from the blue crab alter in vitro calcium carbonate mineralization

Extracellular proteins have long been implicated in the control of mineralization. Cuticular proteins of the blue crab, Callinectes sapidus, undergo changes concurrent with mineralization after molting. A pH drift in vitro mineralization assay was used to assess the effects of cuticular extracts on the inhibition of crystal deposition and the morphology of the resulting crystals. Crystal nucleation was strongly delayed by material extracted into 2% acetic acid within the first 1.5 h post-ecdysis. This effect was not seen with other extracts. No consistent effects of the cuticular extracts on calcium carbonate crystal morphology were observed. Proteins associating with these crystals were extracted and subjected to electrophoresis. These crystal-associated proteins (CAPs) differed depending upon the extraction method and the time after ecdysis when the crab was sacrificed. The early acetic acid extracts produced a unique combination of CAP bands at 66, 42 and 24 kDa. These results are consistent with a model of mineralization in C. sapidus involving (a) certain proteins serving as nucleation sites and attached to the cuticle at all times by acid labile bonds, and (b) other proteins acting as inhibitors of crystal nucleation until being altered at approximately 1 h post-ecdysis.

A THEORETICAL MODEL OF CIRCULATORY INTERSTITIAL FLUID FLOW AND SPECIES TRANSPORT WITHIN POROUS CORTICAL BONE

A three-dimensional model of interstitial fluid flow and passive species transport within mineralized regions surrounding cross-cortical vessel canals is developed. In contrast to earlier studies, the present model applies to circulatory, non-stress-induced interstitial flow in porous cortical bone. Based on previous experimental observations, the canals are modeled as line sources that pass at an oblique angle through the cortex. Cross-cortical interstitial flow from the endosteal surface to the periosteal surface is also taken into account. It is found that model transport characteristics are qualitatively consistent with reported observations. In addition, parametric studies reveal the following: (1) Solute contact with the matrix is maximized when the ratio of canal radius to cortex thickness (R) is near physiological R values. (2) Solute-matrix contact falls to low levels when R falls below the physiological range. (3) Solute-matrix contact is maximized when the cross-cortical velocity is approximately an order of magnitude smaller than the canal outflow velocity. The first and second findings suggest that within porous bone physiological ranges of R promote near optimal species contact with the mineralized matrix. The third finding suggests that relatively impermeable layers of bone within the cortex can effectively promote solute-matrix contact by limiting cross-cortical flow. Finally, the model suggests that intra-canal resorption associated with reduced external loading may effectively compensate for reduced stress-induced interstitial flow by enhancing circulatory interstitial flow and species transport.

Postecdysial changes in the protein and glycoprotein composition of the cuticle of the blue crab Callinectes sapidus

ABSTRACT Postecdysial modifications of the cuticle of Callinectes sapidus were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of soluble protein extracts. Gels stained with Coomassie blue and with silver demonstrated that major changes in protein composition or mobility occurred immediately after ecdysis. Electroblots of SDS-PAGE gels were probed with various biotinylated lectins. The patterns of lectin binding were also markedly different between blots of pre- and postecdysial cuticle extracts, demonstrating a change in glycosylation or glycoprotein mobility in the early postecdysial period. The dramatic alteration in cuticular composition is concurrent with the onset of sclerotization and mineralization of the preexuvially deposited layers of the cuticle.

Lectin Binding by Crustacean Cuticle: The Cuticle of Callinectes Sapidus Throughout the Molt Cycle, and the Intermolt Cuticle of Procambarus Clarkii and Ocypode Quadrata

ABSTRACT Pieces of dorsal carapace from the crayfish Procambarus clarkii, the ghost crab Ocypode quadrata, and the blue crab Callinectes sapidus were fixed in Rossmans fluid or alcoholic Formalin, decalcified, paraffin-embedded, and probed with up to 21 fluorescein-conjugated lectins in order to determine the composition and distribution of their carbohydrate moieties. Unfixed, frozen sections were also employed. Tissue was examined from the crayfish and the ghost crab at intermolt (C4), and from the blue crab at premolt (all D stages), ecdysis, postmolt (all A and B stages, and stage C,) and intermolt. Different lectins showed different binding patterns at intermolt, but patterns of binding for individual lectins were consistent among the different species. A number of lectins showed different binding patterns at different stages of the molt cycle. Lectin binding was influenced by the method of fixation, with Rossmans-fixed tissue binding more lectins and more intensely. The overall pattern of lectin binding indicated that different carbohydrate moieties exist in the different layers of the integument, and that the availability of these lectin-binding sites may change over the molt cycle.

A serum substitute promotes osteoblast-like phenotypic expression in cultured cells from chick calvariae

The effects of medium supplements were tested on embryonic chick calvarial cells in culture. Isolates were divided among four treatment groups: Nu-Serum, chicken serum, fetal bovine serum, or calf serum. Expression of the osteoblastic phenotype was assessed by cell morphology, DNA content, [3H]thymidine incorporation, lactate production, cellular and medium alkaline phosphatase activities, and collagen synthesis. Cells grown in Nu-Serum demonstrated increased alkaline phosphatase activity and a six-fold higher rate of collagen synthesis compared to chicken serum. These cells displayed a polygonal profile, abundant rough endoplasmic reticulum, Golgi apparati, and elaborated an extensive matrix of banded collagen which was well mineralized by day 10 of culture. Although highly mitogenic, chicken serum promoted a more fibroblastoid morphology. Compared to the sera tested, Nu-Serum preferentially promoted the osteoblast-like phenotype in chick calvarial cells in culture.

Structure, molting, and mineralization of the dorsal ossicle complex in the gastric mill of the blue crab, Callinectes sapidus

This study examined the mesocardiac and urocardiac ossicles in the gastric mill of the blue crab to describe its structure, mineralization, and dynamics throughout the molt cycle, and to assess its possible utility in age determination. Morphologically, the mineralized ossicles are similar to the calcified dorsal carapace having a lamellate structure comprised of sheets of chitin/protein fibrils. Staining with acridine orange showed the same arrangement of an epicuticle, exocuticle, and endocuticle. In much of the mesocardiac and urocardiac ossicles, the endocuticle is very reduced, with the exocuticle predominating; the reverse of the dimensions of the exoskeleton. The lamellate structure of the ossicles was confirmed with scanning electron microscopy; however, elemental mapping by energy‐dispersive analysis of X‐rays revealed that the ossicles are mineralized with calcium phosphate, in contrast to the calcium carbonate biomineral of the exoskeleton. The medial tooth of the urocardiac ossicle is not calcified, but the epicuticle is highly elaborated and impregnated with silica. Histological examination of the ossicles demonstrated that they are molted during ecdysis, so despite the appearance of bands in the mesocardiac ossicle, it is difficult to hypothesize how the bands could represent a record of chronological age. J. Morphol. 276:1358–1367, 2015.

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.

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(++).

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.