Continuous culture of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis, and characterization of its morphology, propagation, and extracellular proteins in vitro

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Ultrastructural study of oogenesis and yolk formation in an opisthobranch mollusc, Runcina

Runcina is a hermaphroditic opisthobranch mollusc of small size. It produces large eggs, rich in yolk substances, and it is thus ideal for studying the mechanisms of vitellogenesis. The rough endoplasmic reticulum and the Golgi apparatus appear to be involved in early build up of yolk precursors. Endocytotic vesicles carrying yolk proteins, taken up from the haemolymph, dominate yolk formation at a later stage. The findings presented in this study enhance the proposition of a dual pathway of auto- and heterosynthesis in yolk formation. It is not found that the yolk bodies in Runcina acquire lysosomal enzymes in order to digest the perivitelline fluid, as has been described for some pulmonates. The importance of the role played by the follicle cells in oocyte development is discussed.     

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.     

The circadian rhythm and photosensitivity of small impulses of the Bulla eye

Summary The eye of the mollusk Bulla gouldiana contains a pacemaker that generates a circadian rhythm in compound action potentials (CAPs) in the optic nerve. In this paper, we present evidence of a second circadian rhythm in the optic nerve of the eye maintained in darkness at 15 °C. This is a rhythm in the frequency of small (10–40 V) neural impulses that occurs about 12 h out-of-phase with the rhythm in CAPs. Typically, the small-spike frequency is at a minimum within an hour of the peak in CAP frequency and is maximal during the subjective night. Like the CAP rhythm, the phase of the small-spike rhythm is determined by the prior light/dark cycle. A rebound in small-spike activity following the end of a light pulse and the presence of photoinhibited impulses in surgically reduced eyes suggests that the cells that generate the small-spikes may be photoreceptors that are inhibited by light. In addition, by using isolated nervous system preparations, we have found that smallspikes occur in the two optic nerves in a one-for-one relationship immediately following a light-to-dark transition. This inter-eye communication may be involved in the coupling of the ocular pacemakers.Abbreviations ASW artificial sea water - BRN basal retinal neuron - CAP compound action potential     

Calcium-binding proteins inAplysia neurons

1. Calcium (Ca)-binding proteins of neuronal ganglia and of single, identified neurons of the marine mollusk, Aplysia californica, were investigated. Using transblot/45Ca overlays two proteins, at Mr 45,000 and Mr 23,000, with a high Ca-binding ability were found. 2. Western blot analysis revealed that the protein at Mr 45,000 could be separated by 2D-PAGE into proteins with Mr 40,000 and Mr 43,000. The protein at Mr 40,000 immunocross-reacted with antisera directed against parvalbumin and rat calbindin D-28K, indicating a novel Ca-binding protein sharing common antigenic determinants for both proteins. 3. The protein at Mr 23,000 could be separated into a group of proteins with Mr 13,000-20,000 which showed a high degree of similarity to sarcoplasmatic calcium-binding proteins (SCP). 4. We further investigated the protein pattern of single, identified neurons of different electrical activity (bursting, beating, and silent) by 2D-PAGE. Major differences were found in the range of low Mr and low pI, where Ca-binding proteins are generally located. A protein at high concentrations characteristic for silent cells migrated at a position similar to crayfish SCP. 5. The results show that various Ca-binding proteins are characteristic for neurons in the Aplysia nervous system and support the idea that they may effect the electrical behavior of nerve cells.     

Complete sequences of the highly rearranged molluscan mitochondrial genomes of the Scaphopod Graptacme eborea and the bivalve Mytilus edulis

We have determined the complete sequence of the mitochondrial genome of the scaphopod mollusk Graptacme eborea (14,492 nts) and completed the sequence of the mitochondrial genome of the bivalve mollusk Mytilus edulis (16,740 nts). (The name Graptacme eborea is a revision of the species formerly known as Dentalium eboreum.) G. eborea mtDNA contains the 37 genes that are typically found and has the genes divided about evenly between the two strands, but M. edulis contains an extra trnM and is missing atp8, and it has all genes on the same strand. Each has a highly rearranged gene order relative to each other and to all other studied mtDNAs. G. eborea mtDNA has almost no strand skew, but the coding strand of M. edulis mtDNA is very rich in G and T. This is reflected in differential codon usage patterns and even in amino acid compositions. G. eborea mtDNA has fewer noncoding nucleotides than any other mtDNA studied to date, with the largest noncoding region only 24 nt long. Phylogenetic analysis using 2,420 aligned amino acid positions of concatenated proteins weakly supports an association of the scaphopod with gastropods to the exclusion of Bivalvia, Cephalopoda, and Polyplacophora, but it is generally unable to convincingly resolve the relationships among major groups of the Lophotrochozoa, in contrast to the good resolution seen for several other major metazoan groups.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

Concentrations of Heavy Metals in Soft Tissues of the Gastropod Nucella heyseanain the Vicinity of Vladivostok and in the Vostok Bay Reserve

A survey was made of 168 specimens of the gastropod mollusk Nucella heyseanafrom 5 samples collected in Amurskii Bay and Vostok Bay (Peter the Great Bay and the Sea of Japan). The variability of Cu, Zn, Cd, Pb, Fe, and Mn contents in gastropod soft tissues was analyzed. The statistic concentrations of the six metals differed significantly among the samples; they were sometimes taken at a distance of a mere several tens of meters apart. N. heyseanacollected near Vladivostok generally had higher concentrations of heavy metals; however, the amount of Zn in the specimens of one sample from the Vostok Bay Reserve was 400 g/g, which is two times greater than that of mollusks from the urbanized zone. The canonical and discriminant analyses and the analysis of the variance indicate that the variation in heavy metal concentration within groups of mollusks (within bays) is comparable in magnitude with the variation among the groups.     

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well‐documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral‐associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post‐translational modifications, sequence redundancy, and amyloid‐like aggregation‐prone sequences. Together, these molecular features create a protein‐based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.