Topology of the 10 subunits within the decamer of KLH,the hemocyanin of the marine gastropod Megathura crenulata

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.     

The effect of captivity and diet on KLH isoform ratios in Megathura crenulata

Aquaculture of the giant keyhole limpet, Megathura crenulata, may provide a reliable long-term supply of keyhole limpet hemocyanin (KLH) for many promising biomedical applications. However, previous studies have reported a complete loss of the KLH1 isoform under certain cultivation conditions. We examined whether captivity per se and diet caused a significant change in the isoform profile of M. crenulata. Although there was a trend toward a decreasing percentage of KLH1 in some animals, in general isoform profiles were not significantly affected by captivity or dietary limitations. Further, the percentage of KLH1 significantly increased for limpets with previously low levels of KLH1 when fed a supplemental mixed diet. Our results indicate that normal isoform profiles can be maintained in limpets held in captivity even when fed insufficient diets, and that these conditions do not cause a complete loss of either KLH isoform. Notably, the enhancement of abnormally low levels of KLH1 suggests that variability in isoform profiles could potentially be minimized through diet. While there is a need for further research on the factors responsible for the variability of KLH, overall, these results support the premise that culture of M. crenulata may provide a sustainable source of this biomedically important product.     

Ultrastructural correlates of interneuronal function in the abdominal ganglion of Aplysia californica

The synaptic inputs and outputs of the major interneuron L10 of the abdominal ganglion of Aplysia were studied using an intracellular staining technique for the electron microscope. The sites of both the chemical synaptic input and output of L10 are localized to the dendritic arborizations that arise from the axon in the ganglion neuropil. Thus, the interneuronal functions are mediated at the dendritic processes and could occur in the absence of spiking in the axon and cell body. The sites of L10 synaptic output are presumed to be at. aggregations of vesicles and mitochondria in the dendrites. The synaptic vesicle content of L10, a cholinergic neuron, with many large dense vesicles resembles that described for serotonergic cells in Aplysia, making distinction of synaptic pharmacology by ultrastructure difficult. Focal membrane specializations with a clear synaptic cleft were not observed between L10 and its large population of postsynaptic cells. In contrast, clear focal input sites were frequently found on L10. Gap junctions, sites of probable electrical coupling between L10 and other neurons, were also found. These observations are discussed as evidence that many synapses do not have focal specializations.     

Cerebrin prohormone processing, distribution and action in Aplysia californica

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.     

The peritrophic membrane of the gastropod Megathura crenulata: structure,composition, and site of formation

Peritrophic membranes (PMs) are acellular layered structures secreted around ingested materials by the gut epithelium. Most studies on PMs have focused on those of insects and crustaceans due to their potential ability to block the movement of pathogens from ingested materials into the body, and their possible use as unique targets relevant to pest management. While PMs are known to occur in other taxa, their distribution is spotty and little is known about their role in these other species. The gastropod Megathura crenulata produces a true PM, which has a chitinous matrix that makes up nearly half its wet weight. Unlike arthropod PMs, which are released by delamination from the microvilli of their gut cells, the chitinous matrix of the M. crenulata PM is secreted from epithelial cells lining most regions of its gut. Although its mode of synthesis is unique, it may serve the same functions as proposed for other PMs, including regulating diffusion, binding metabolites, restricting protease activity, blocking pathogens, and providing lubrication. In arthropods, numerous proteins with chitin‐binding specificities have been identified, consistent with the proposed functions. Analysis of PMs in M. crenulata showed several integral proteins associated with the membrane, suggesting that the PM in this mollusc may be involved in complex functions like those seen in the arthropods.     

Ultrastructural correlates of interneuronal function in the abdominal ganglion of Aplysia californica.

The synaptic inputs and outputs of the major interneuron L10 of the abdominal ganglion of Aplysia were studied using an intracellular staining technique for the electron microscope. The sites of both the chemical synaptic input and output of L10 are localized to the dendritic arborizations that arise from the axon in the ganglion neuropil. Thus, the interneuronal functions are mediated at the dendritic processes and could occur in the absence of spiking in the axon and cell body. The sites of L10 synaptic output are presumed to be at aggregations of vesicles and mitochondria in the dendrites. The synaptic vesicle content of L10, a cholinergic neuron, with many large dense vesicles resembles that described for serotonergic cells in Aplysia, making distinction of synaptic pharmacology by ultrastructure difficult. Focal membrane specializations with a clear synaptic cleft were not observed between L10 and its large population of postsynaptic cells. In contrast, clear focal input sites were frequently found on L10. Gap junctions, sites of probable electrical coupling between L10 and other neurons, were also found. These observations are discussed as evidence that many synapses do not have focal specializations.     

cDNA Sequence, Protein Structure, and Evolution of the Single Hemocyanin from Aplysia californica, an Opisthobranch Gastropod

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5UTR of 77 bp, a 3UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (M_r ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (M_r ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373±47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343±43 million years ago.[Reviewing Editor: Dr. Axel Meyer]The sequence reported in this paper has been deposited in the GenBank database under accession number AJ556169.     

Specific, water-soluble polypeptides in identified neurons of Aplysia californica.

Application of an ethylene glycol lysis technique to extract water-soluble, low molecular weight polypeptides in Aplysia neurons, was used in conjunction with microgradient gel electrophoresis and micro-isoelectric focusing, to identify unique polypeptides in specific, identified neurons. The polypeptides found in neurons R15, R3-13, R14, and the bag cells were particularly abundant, consistent with the previously suggested neurosecretory role for these cells. Water extraction of the strongly basic polypeptides (pI 10.7) in R3-13 and R14 required an acidic lysis medium.     

Characterization of Neuronal Protein Phosphatases in Aplysia californica

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.     

Choline acetyltransferase in individual neurons of Aplysia californica

The activities of choline acetyltransferase in the various ganglia of the nervous system of Aplysia californica and in some of the individually identifiable neurons in these ganglia were measured. At least four of the neurons were characterized by an apparent absence of the enzyme. The neurons containing measurable amounts of the enzyme had reproducible levels from animal to animal. Individual neurons from the same animal were generally characterized by different levels of activity whether expressed on a cell or a protein basis. However, those pairs of neurons previously classified as ‘homologous’ because of their similar appearance, location and/or electrophysiological function, also contained the same total amounts of enzyme activity.     

Stages in the post-hatching development of Aplysia californica

In order to study the development of the nervous system of the marine mollusc, Aplysia californica, it is necessary objectively to assess the maturity of individual specimens. This can be done by defining stages in the life cycle. The post-hatching development can be divided into four phases: planktonic, metamorphic, juvenile, and adult. These phases can be further subdivided into 13 stages on the basis of behavioral and morphological characteristics visible in living specimens: Stage 1, newly hatched; Stage 2, eyes develop; Stage 3, the larval heart beats; Stage 4, maximum shell size is reached; Stage 5, the propodium develops; Stage 6, red spots appear; Stage 7, the velum is shed; Stage 8, eyebrows appear; Stage 9, pink color develops; Stage 10, white spots appear; Stage 11, rhinophores grow; Stage 12, the genital groove forms; Stage 13, egg laying begins. Reconstructions from serial sections taken from specimens fixed at each of these stages reveal the sequence of formation of the major organ systems. The nervous system develops gradually. The cerebral and pedal ganglia are present at Stage 1, the optic ganglia develop at Stage 2, the abdominal, pleural, and osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and the genital ganglion at Stage 13. Because Aplysia develops gradually, it is possible to analyze the contribution which gastropod torsion makes to the different phases of the life cycle. The Aplysia embryo undergoes 120 degrees torsion prior to Stage 1. The major visceral organs, the digestive system, heart, gill, and visceral nervous system, develop sybsequently in their post-torsional positions. After metamorphosis, there is a partial de-torsion which involves only the digestive system. Torsion of the digestive system may therefore be beneficial only to the pre-metamorphic larva, and not to the postmetamorphic juvenile.     

Electron microscopy of a double helical tubular filament in keyhole limpet (Megathura crenulata) hemolymph

Summary A 25 nm hollow double helical filament has been detected ultrastructurally in the cell-free supernatant from hemolymph of the keyhole limpet Megathura crenulata (Gastropoda: Prosobranchia: Fissurellidae). Subsequently, much higher concentrations of this material were found in the cell pellet from hemolymph. Both negative staining and thin sectioning have been performed in an attempt to obtain a preliminary structural characterization of this new filament. It is proposed that the filaments are released or secreted from blood hemocytes in response to bleeding, but it has not been possible to define absolutely an intracellular organelle containing this material. It is shown that the hollow filaments, which have fine fibrillar surface extensions, are readily distinguishable from collagen fibrils and from tubular (multi-decameric) keyhole limpet hemocyanin (KLH).     

Neuroendocrine Regulation of Egg Laying in Aplysia californica

Two clusters of neurons, the bag cells, associated with thecentral nervous system of Aplysia californica play an essentialrole in the induction of egg laying by the animal. Studies concernedwith the morphology, electrophysiology, biochemistry, and functionof these cells are reviewed and discussed. The unusually favorablecharacteristics of this preparation suit it for developmentas a model neuroendocrine effector system.     

Mechanical reconfiguration mediates swallowing and rejection in Aplysia californica

Muscular hydrostats, such as tongues, trunks or tentacles, have fewer constraints on their degrees of freedom than musculoskeletal systems, so changes in a structure’s shape may alter the positions and lengths of other components (i.e., induce mechanical reconfiguration). We studied mechanical reconfiguration during rejection and swallowing in the marine mollusk Aplysia californica. During rejection, inedible material is pushed out of an animal’s buccal cavity. The grasper (radula/odontophore) closes on inedible material, and then a posterior muscle, I2, pushes the grasper toward the jaws (protracts it). After the material is released, an anterior muscle complex (the I1/I3/jaw complex) pushes the grasper toward the esophagus (retracts it). During swallowing, the grasper is protracted open, and then retracts closed, pulling in food. Grasper closure changes its shape. Magnetic resonance images show that grasper closure lengthens I2. A kinetic model quantified the changes in the ability of I2 and I1/I3 to exert force as grasper shape changed. Grasper closure increases I2’s ability to protract during rejection, and increases I1/I3’s ability to retract during swallowing. Motor neurons controlling radular closure may therefore affect the behavioral outputs of I2’s and I1/I3’s motor neurons. Thus, motor neurons may modulate the outputs of other motor neurons through mechanical reconfiguration.Valerie A. Novakovic and Gregory P. Sutton contributed equally to the paper.