Length-dependent deactivation of ventricular trabeculae in the bivalve, Spisula solidissima

Shortening-deactivation has been identified and characterized in ventricular trabeculae of the bivalve, Spisula solidissima (Heterodonta, Mactridae). This muscle had ultrastructural similarities to vertebrate smooth muscle. Deactivation was defined as the fraction of maximal force lost during a contraction when a muscle is shortened rapidly (by a quick-release, QR) to a known length, relative to a control isometric contraction at that same length. The magnitude of deactivation was dependent on the size of the release and the point at which the release was applied during the cycle of contraction. QR/quick-stretch (QS) perturbations at the same point during the contraction resulted in negligible deactivation. The magnitude of deactivation was independent of shortening rate. Deactivation was attenuated by applying caffeine (100 μM) and blocked with high extracellular Ca²⁺ (56 mM). The Ca²⁺ ionophore, A23187 (10 μM), augmented deactivation as did the positive inotrope serotonin (100 nM). Treatment with ryanodine (5 μM) had no significant effect on deactivation. These results suggest that a reduction in Ca²⁺ at the contractile element and/or sequestration of Ca²⁺ may occur during shortening. Deactivation may minimize the magnitude of work done during active shortening of bivalve cardiac muscle, particularly against the low afterload exhibited in the bivalve peripheral circulatory system. Intracellular Ca²⁺ fluxes during sudden length perturbations may explain the effect of stretch on action potential duration in the bivalve heart, as shown previously.     

Aurora B Kinase Maintains Chromatin Organization During the MI to MII Transition in Surf Clam Oocytes

Meiosis represents a specialized cell cycle whereby cells undergo two reductive divisions without an intervening S phase. In oocytes, the transition from meiosis I to II is brief, with paired sister chromatids remaining condensed throughout the interkinesis period. This stands in contrast to mitotic divisions where cytokinesis and the return to interphase is always accompanied by chromatin decondensation and nuclear envelope reformation. Because other aspects of M phase exit are normal, we probed the mechanisms that allow for polar body extrusion while retaining chromatin condensation in Spisula solidissima oocytes. If oocytes were activated in the presence of protein synthesis inhibitors, oocytes progressed normally through MI, but arrested in interkinesis with condensed chromatin, phosphorylated histone H3 and a disorganized MII spindle. Neither inhibition of CDK1- nor MAPK activity in arrested oocytes was sufficient to drive chromatin decondensation or nuclear envelope reformation, suggesting that these kinases were not responsible for the maintenance of chromatin condensation. However, inhibition of Aurora B kinase activity resulted in chromatin decondensation, loss of histone H3 phosphorylation and reformation of the nuclear envelope. Inhibition of Aurora B activity following MI also resulted in chromosome segregation defects during MII and blocked polar body formation, consistent with Aurora B’s well-established role in cytokinesis. Together, these results suggest that extended Aurora B activity between meiotic divisions maintains chromatin condensation, thus allowing for the rapid reassembly of the MII spindle and progression through meiosis.     

Localization and quantification of gonad serotonin during gametogenesis of the surf clam, Spisula solidissima

In the surf clam, Spisula solidissima, serotonin was reported to induce spawning when injected into the gonads. At nanomolar concentrations, it facilitates the fertilizability of oocyte by sperm, at micromolar concentration, it triggers the meiotic maturation of prophase 1-arrested oocytes, thus mimicking the effect of sperm. To further understand the role of serotonin in the gametogenic and spawning processes, we used both immunohistochemistry and high-pressure liquid chromatography linked with electrochemical detection to detect serotonin in the gonads of the surf clam. We found serotonin-containing varicose fibers covering the surface of the germinal epithelium in both sexes. The area occupied by the serotonergic innervation field encircling gonad acini varied according to the gonadal stages (active phase, ripe phase, partially spawned phase, spent phase). We also found large variations in the serotonin concentration between specimens during the gametogenic cycle. The serotonin concentration was correlated with gonad growth: it decreased in the ripe phase in comparison with the previous phase, the active phase. We attribute the decrease to the increase of total gonad mass in this stage. In contrast, as spawning begins, the total gonad mass declines while the gonad serotonin concentration increases to a level similar to that found in active phase. The finding that prior to spawning, serotonin is present in the gonads within fibers exhibiting distinct varicosities suggests that it is implicated in spawning.     

Temporal and spatial expression of embryonic alkaline phosphatase activity in the surf clam,Spisula solidissima

The temporal appearance and spatial distribution of alkaline phosphatase (EC 3.1.3.1) have been examined during the development of the surf clam, Spisula solidissima. Enzyme activity was detectable at low levels from fertilization onward with a sharp linear increase in activity occurring at the trochophore stage, 14 h post-fertilization. Histochemical localization of enzyme activity at the light microscope level showed the increased activity associates initially with a limited number of large macromeres and is progressively restricted to the cells forming the tip of the growing archenteron. Electron microscopy shows the activity is restricted to the luminal and lateral plasma membrane of these cells.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.     

The relationships between early ionic events, the pattern of protein synthesis, and oocyte activation in the surf clam, Spisula solidissima

The ionic events linked to activation of surf clam (Spisula solidissima) oocytes include a transient increased Ca2+ influx and an acid release. The aim of the present work was to further elucidate the respective roles of these two ionic events and to clarify the possible role of protein kinase C in the sequence of events leading to oocyte activation. K+-enriched seawater, ammonium chloride, and the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, were tested for their ability to promote germinal vesicle breakdown (GVBD), an acid release, increased 45Ca2+ uptake, and a shift in the pattern of protein synthesis. Oocytes activated by addition of K+ ions release an amount of H+ similar to that induced by fertilization, with the same time course, show an increased, verapamil-sensitive, 45Ca2+ uptake that is proportional to the amount of added K+, and undergo a shift in their pattern of protein synthesis, which requires the presence of external Ca2+. Ammonium chloride, at concentrations causing a higher production of acid than that induced by K+ ions or fertilization, does not trigger GVBD nor any increased 45Ca2+ uptake or any detectable shift in the pattern of protein synthesis. Combined additions of ammonium chloride with subthreshold concentrations of K+ ions allow GVBD to occur, thus revealing a synergistic effect of ammonia and K+ ions. TPA slowly induces GVBD, an Na+-dependent acid release, and a shift in the pattern of protein synthesis, in the absence of increased 45Ca2+ uptake. Our results lead us to propose the following sequence of events for the activation of Spisula oocytes: an increased Ca2+ influx contributes to activate protein kinase C which causes a Na+-dependent acid release leading to a rise of pHi. This rise of pHi, although insufficient by itself, may set the pHi in a permissive range for activation to occur through the action of other protein kinase C-sensitive events leading to the production of meiosis-inducing proteins.     

Additive interactions between the moon snail Euspira heros and the sea star Asterias forbesi, two predators of the surfclam Spisula solidissima

A laboratory experiment was conducted to determine whether the sea star Asterias forbesi and the naticid gastropod Euspira heros feed on surfclams, Spisula solidissima, in an additive or non-additive manner. Predators were allowed to feed on clams with conspecifics and in the presence of the other predator species. Clam mortality (measured as the rate of decline of clam number) and predator feeding rates were noted. To determine the effects of temperature on interactions among the predators, the experiment was conducted at three different temperatures. At all temperatures, feeding rate of each predator was not affected by the presence of the other species, and clam mortality in the presence of both predators was predictable from mortality in the presence of a single predator species. These additive interactions are most likely a result of habitat partitioning between the predators, with naticid snails being infaunal and sea stars being epifaunal. Previous studies in a variety of systems show no clear pattern of occurrence of non-additive interactions. Relatively small differences in predator or prey behavior may be responsible for the presence or absence of non-additive interactions. Received: 6 August 1998 / Accepted: 25 January 1999     

Studies on Shell Formation : VII. The Submicroscopic Structure of the Shell of the Oyster Crassostrea virginica

The submicroscopic structure of the growing surface of the shell of the oyster, Crassostrea virginica, was studied by means of shadowed replicas. The outer edge of the prismatic region consists of a fine grained matrix enclosing crystals, the surfaces of which show a finely pebbled structure. Crystal size varies continously from 0.01 µ to 8 µ. The matrix surface shows no evidence of fibrous structure. The outer portions of the prismatic region exhibit a tile-like arrangement of large crystals separated by granular matrix 0.02 to 0.08 µ in thickness. The exposed crystal surfaces have indentations of varying form which appear as roughly parallel grooves spaced at intervals of approximately 0.3 µ. The final form of this region is believed to result from the random distribution of crystal seeds, which grow without orientation and through coalescence and growth come into contact, producing polygonal areas. The crystal arrangement of the nacreous region is one of overlapping rows of crystals in side to side contact, and with one end of each crystal free, permitting continued increase in length. Crystal angles and plane indices are presented.     

Developmental Patterns in Histones and Histone Synthesis During Early Embryogenesis of the Sea Urchin Paracentrotus lividus

Abstract. Striking developmental changes in histone and histone synthesis in sea urchin embryos were observed in three histone classes, H1, H2A and H2B. In each case there is a shift in histone synthesis from the early cleavage stage types to other types of histones at the morula stage; Two new forms appear after the blastula stage. In addition, multiple changes in histone types were found during gameto-genesis in the male and female gonads where specific histones, different from the embryonic histones, were observed.     

Growth history and ecology of the Atlantic surf clam,Spisula solidissima (Dillwyn), as revealed by stable isotopes and annual shell increments

The shells of the Atlantic surf clam, Spisula solidissima (Dillwyn), contain a record of both life history and environmental changes. These shell records were investigated using oxygen and carbon stable isotopic analyses (δ¹⁸O, δ¹³C) and shell growth increment analyses. δ¹⁸O variations across annual shell increments reflect the yearly cycle of sea-water temperatures off the New Jersey coast, further documenting the proposed annual periodicity of the major shell increments. The 11-yr shell record analyzed here confirms that shell growth is most rapid in spring-early summer, slow in late summer-fall, and extremely slow or non-existent in winter. Shell growth appears to occur in isotopic equilibrium with sea water and measured δ¹⁸O values are used to refine the aragonite-water temperature scale. Variations in the timing of annual growth increment formation are noted as well as ontogenetic effects upon the range of isotopic values recorded in shell carbonate. Both the δ¹⁸O and δ¹³C profiles are influenced by changes in the sea-water temperature regime over the 11-yr period studied (1965–1976) and record these effects in the shell. The combination of stable isotope and growth increment analyses provides a powerful tool for interpreting the shell records of both modern and fossil molluscs.     

Early Embryogenesis and Anterior-Posterior Axis Formation in the White-Tip Nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae)

We followed the early embryogenesis of Aphelenchoides besseyi from fertilization to the 4-cell stage under Nomarski optics and examined the chromosome number and structure by DAPI staining. After an oocyte is fertilized by a sperm, the eggshell forms and the male and female pronuclei are reconstructed. The male pronucleus moves toward the female pronucleus, which is located at the center of the egg. They meet, rotate 90°, and fuse. The embryo then divides unequally into a larger anterior AB cell and a smaller posterior P(1) cell. The site of sperm entry into the oocyte seems to become the future anterior pole of the embryo, and thus the formation of an anterior-posterior axis formation is the same as that for Bursaphelenchus xylophilus, but opposite to that for Caenorhabditis elegans. From immunostaining, the fertilizing sperm appears to bring the centrosome into the oocyte. The chromosome structure during the pronuclear meeting as observed by DAPI staining suggests that a haploid sperm (N = 3) fertilizes a haploid oocyte (N = 3) to form a diploid embryo (2N = 6) and that all chromosomes appear to be of a similar size. Unlike C. elegans does, the P(1) cell first divides anterior-posteriorly followed by the AB anterior-posteriorly. These divisions produced the 4-cell stage embryo with 4 cells arranged in a linear fashion, again in contrast to that for C. elegans or B. xylophilus configured in a rhomboid shape.