Use of monoclonal antibodies for identification of growth-controlling neuropeptides in the mussel Mytilus edulis (Mollusca: Bivalvia)

Monoclonal antibodies were developed against cerebral ganglia (CG) of the mussel Mytilus edulis by the immunization of mice with unpurified homogenates of these organs. The screening protocol of hybridoma was based upon immunohistological observations of cytocentrifugated ganglia cells. A panel of 29 monoclonal antibodies (MABs) specific of CG epitopes was harvested and subsequently used for the immunocytochemical study of CG cells. Several subpopulations of ganglia cells were specifically revealed by MABs. Identification of epitopes involved in growth control was approached via the application of a bioassay allowing the assessment of protein synthesis stimulation. MAB 42 and 46 affected amino acid incorporation induced by CG extract. These results lead to the conclusion that the epitopes recognised by these antibodies are involved in growth control. An immunoenzymatic assay was performed with CG extracts for quantitative analyses of epitopes.     

A comparison of relative growth in Cerastoderma (=Cardium) edule, Modiolus modiolus, and Mytilus edulis (Mollusca: Bivalvia)

Relative growth has been studied in intertidal populations of Cerastoderma edule and Mytilus edulis and in a subtidal population of Modiolus modiolus. Size dimensions were generally more variable and shell thickness generally greater in the intertidal species. Total weight was correlated with shell weight in all three species but there was a progressively greater emphasis on tissue growth through the sequence Cerastoderma, Mytilus, Modiolus. These results are interpreted in terms of differences in stability between their respective habitats.
Analyses of individual year classes in Cerastoderma showed a marked pattern of alternating years of good and poor growth.     

Mytilus galloprovincialis (Mollusca: Bivalvia) in a warm-temperate South African estuarine embayment

The distribution, population structure, condition and gonad indices, and the attachment strengths of the invasive Mediterranean mussel Mytilus galloprovincialis are described from the Knysna Estuary, South Africa. Mussels were found in the rocky intertidal at the mouth of the estuary and on all man-made hard substrata as far as 12?km from the mouth in 2013. However, mussel beds were found only in the lower reach of the estuary, where mussel densities were as high as 120 per 0.01?m². When compared to mussels from the embayment regions of the estuary, those at the estuary mouth, where wave action was high, were generally smaller, had significantly lower condition and gonad indices, and greater attachment strength. This suggests that mussels within the sheltered regions of the estuary invest more energy in growth and reproduction, and that this is one reason for their success as an invasive species in the Knysna estuarine embayment.     

Mitochondrial DNA polymorphisms and sperm motility in Mytilus edulis (Bivalvia: Mytilidae)

The system of mitochondrial DNA (mtDNA) inheritance in Mytilus and other bivalves, termed doubly uniparental inheritance (DUI), is novel among animals. Males pass on their male transmitted (M-type) mtDNA from fathers to their sons whereas females pass on their female transmitted (F-type) mtDNA from mothers to both sons and daughters. Thus, Mytilus males contain two distinct types of mtDNA. Interestingly, sperm contains only the paternal mtDNA. Phylogenetic analysis has shown that some female types have been able to switch their route of inheritance. These "recently masculinized" mitochondrial genomes behave as a typical M-type in that they are transmitted from generation to generation through sperm. Because the "recently masculinized" and "standard" male mitotypes in M. edulis exhibit approximately 8.7% amino acid sequence divergence, we hypothesized that these differences could affect mitochondrial, and hence sperm, functions. Furthermore, since recently masculinized mitotypes have been shown to replace standard male types periodically over evolutionary timescales, we tested the hypothesis that sperm swimming speeds would be greater for males with recently masculinized M-type genomes. Sperm activity was videotaped, digitized and tracked. A linear mixed effects model found no significant difference in linear velocities or curvilinear speeds between the mitotypes suggesting that swimming speeds are similar for both in the period shortly after spawning.     

Feeding and digestion by the mussel Mytilus edulis L. (Bivalvia: Mollusca) in mixtures of silt and algal cells at low concentrations

Mussels Mytilus edulis L. from two populations were exposed for 2 days or 2 wk to mixtures of silt and the diatom Phaeodactylum tricomutum Bohlin before measuring rates of feeding, the passage time for food in the gut, absorption efficiency and metabolic rate. Experimental diets were set up to span the range of organic content in seston from the natural habitats (% organic matter by weight: 7–55%), but were less than the total levels of natural seston. Absorption efficiencies were adequately modelled in an exponential relationship to food quality and to gut passage time, although at high proportional silt concentrations metabolic faecal losses led to negative net rates of absorption. Over short-term exposures the scope for growth was a simple function of food quality, and the nutritional quality of the diet was best expressed as organic content per unit volume of particles. Over a period of 2 wk physiological acclimation occurred, across all levels of experimental food quality (which were as low as 10% organic matter by weight), resulting in positive growth potential. Relevant mechanisms of compensation include increased rates of ingestion, increased absorption efficiency and an apparent increase in digestive capacity, estimated here as gut fullness. In experiments in which natural diets are simulated by adding silt to phytoplankton cells, the consequences for net rates of absorption depend on the balance between mean particle size and organic content per unit volume. Calculations show how, in some circumstances, growth may be enhanced by the addition of small silt particles to living phytoplankton cells.     

Muscle and neuronal differentiation in primary cell culture of larval Mytilus trossulus (Mollusca: Bivalvia)

Molluscan in vitro technology allows the study of the differentiation of isolated cells undergoing experimental manipulations. We have used the immunofluorescence technique and laser scanning microscopy to investigate the organization of muscle proteins (actin, myosin, paramyosin, and twitchin) and the localization of neurotransmitters (serotonin and FMRFamide) in cultured mussel larval cells. Differentiation into muscle and neuron-like cells occurs during the cultivation of mussel cells from premyogenic and prenervous larval stages. Muscle proteins are colocalized in contractile cells through all stages of cultivation. The cultivation of mussel cells on various substrates and the application of integrin receptor blockers suggest that an integrin-dependent mechanism is involved in cell adhesion and differentiation. Dissociated mussel cells aggregate and become self-organized in culture. After 20 days of cultivation, they form colonies in which serotonin- and FMRFamide-immunoreactive cells are located centrally, whereas muscle cells form a contractile network at the periphery. The pattern of thick and thin filaments in cultivated mussel cells changes according to the scenario of muscle arrangement in vivo: initially, a striated pattern of muscle filaments forms but is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins, typical of muscles of adult molluscs. Myogenesis in molluscs thus seems to be a highly dynamic and potentially variable process. Such a “flexible” developmental program can be regarded as a prerequisite for the evolution of the wide variety of striated and smooth muscles in larval and adult molluscs.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Expression of thick filament proteins during ontogenesis of the mussel Mytilus trossulus (Mollusca: Bivalvia)

The appearance of thick filament proteins organized into supramolecular complexes was studied by SDS-PAGE and Western-blot analysis at different developmental stages of the mussel Mytilus trossulus. Paramyosin appeared at the egg stage, while twitchin and myorod appeared at the blastula stage (12 h after fertilization). In addition, RT-PCR analysis showed that the twitchin genes were expressed starting from the blastula stage. Thus, the proteins forming thick filaments of the contractile apparatus of mussel muscles are expressed long before the formation of the first well-organized muscle system of the veliger larvae (55 h). Further, the ratios actin/myosin heavy chain (MHC) and paramyosin/MHC at the veliger stage (96 h) distinctly differed from those in the adult mussel.     

Tissue Hemoglobins in Bivalvia (Mollusca)

Distribution and properties of tissue hemoglobins in bivalves are reviewed. The mollusk gills are given particular attention; data on their functions, relative weight, and surface area as well as the content, properties, and functional importance of gill hemoglobin are reviewed.     

Modulation of Mytilus trossulus (Bivalvia: Mollusca) larval survival and growth in culture. Short communication

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.     

Geometrical and crystallographic constraints determine the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca)

Unionid shells are characterized by an outer aragonitic prismatic layer and an inner nacreous layer. The prisms of the outer shell layer are composed of single-crystal fibres radiating from spheruliths. During prism development, fibres progressively recline to the growth front. There is competition between prisms, leading to the selection of bigger, evenly sized prisms. A new model explains this competition process between prisms, using fibres as elementary units of competition. Scanning electron microscopy and X-ray texture analysis show that, during prism growth, fibres become progressively orientated with their three crystallographic axes aligned, which results from geometric constraints and space limitations. Interestingly transition to the nacreous layer does not occur until a high degree of orientation of fibres is attained. There is no selection of crystal orientation in the nacreous layer and, as a result, the preferential orientation of crystals deteriorates. Deterioration of crystal orientation is most probably due to accumulation of errors as the epitaxial growth is suppressed by thick or continuous organic coats on some nacre crystals. In conclusion, the microstructural arrangement of the unionid shell is, to a large extent, self-organized with the main constraints being crystallographic and geometrical laws.     

An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male)

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.     

A comparison of variability in size dimensions of intertidal and subtidal mussels (Mytilus edulis) (Mollusca: Bivahia)

Variability in size dimensions of bivalve species has previously been claimed (1) to decrease with size and (2) to be greater for individuals in an intertidal habitat than in a subtidal habitat. Results from this study showed that the decrease in variability with size was an artifact of the techniques previously used, and that variability was the same for all sizes of Mytilus edulis. The previous study utilized an interspecific comparison to examine the effects of habitat on variability, which is considered unacceptable because differences in variability are likely to be a consequence of species differences rather than the effects of different habitats. Results presented here from the intraspecific comparison of M. edulis showed that variability was the same for mussels from an intertidal habitat and from a subtidal habitat.     

A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca)

The periostracum in Unionidae consists of two layers. The outer one is secreted within the periostracal groove, while the inner layer is secreted by the epithelium of the outer mantle fold. The periostracum reaches its maximum thickness at the shell edge, where it reflects onto the shell surface. Biomineralization begins within the inner periostracum as fibrous spheruliths, which grow towards the shell interior, coalesce and compete mutually, originating the aragonitic outer prismatic shell layer. Prisms are fibrous polycrystalline aggregates. Internal growth lines indicate that their growth front is limited by the mantle surface. Transition to nacre is gradual. The first nacreous tablets grow by epitaxy onto the distal ends of prism fibres. Later growth proceeds onto previously deposited tablets. Our model involves two alternative stages. During active shell secretion, the mantle edge extends to fill the extrapallial space and the periostracal conveyor belt switches on, with the consequential secretion of periostracum and shell. During periods of inactivity, only the outer periostracum is secreted; this forms folds at the exit of the periostracal groove, leaving high-rank growth lines. Layers of inner periostracum are added occasionally to the shell interior during prolonged periods of inactivity in which the mantle is retracted.     

Extracellular matrix is required for muscle differentiation in primary cell cultures of larval Mytilus trossulus (Mollusca: Bivalvia)

Components of the extracellular matrix may modulate the growth factor effects that play important roles in the proliferation and differentiation of precursor cells. We developed an in vitro cultivation protocol for cells of the larval marine bivalve Mytilus trossulus to study the role that extracellular matrix components may play in myodifferentiation and replication-mediated DNA synthesis using immunofluorescence and confocal laser scanning microscopy. Here, we demonstrate that the extracellular matrix regulates the expression of muscle proteins, leading to their assembly and the terminal muscle differentiation of larval cells during cultivation. We further show that the myogenesis process progresses in cells cultivated on fibronectin, carbon or poly-l-lysine but is inhibited in cells grown on a collagen carpet. Consistent with a decrease in muscle protein expression in cells cultivated on collagen, we demonstrate an increase in the number of BrdU-positive cells in comparison with cells cultured on other substrates during the entire cultivation period. Moreover, we demonstrate that the matrix-dependent myogenic differentiation of larval mussel cells is reversible. Round-shaped cells cultivated on collagen were able to differentiate into muscle cells after reseeding on fibronectin, carbon or poly-l-lysine. In addition, cells cultured on collagen and then transplanted to fibronectin exhibited distinct cross-striation and contractile activity. Taken together, our data suggest that the extracellular matrix participates in the regulation of the proliferation and myodifferentiation of mussel trochophore progenitor cells and validate novel approaches for successfully culturing cells from bivalves over extended periods.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

Enkephalin-like immunoreactivity in the pedal ganglion of Mytilus edulis (Bivalvia) and its proximity to dopamine-containing structures

The presence of enkephalin-like immunoreactivity in the pedal ganglion of the bivalve mollusc Mytilus edulis has been demonstrated. Enkephalin-like immunoreactivity is highly localized in those intraganglionic regions rich in dopamine-containing structures. In addition, FMRF-NH2-like (Phe-Met-Arg-Phe-HN2) immunoreactivity was found to occur in intraganglionic regions that are devoid of enkephalin-like immunoreactivity. The differential topographic distribution of these peptidergic immunoreactivities suggests involvement in separate functions. By contrast, the closeness of enkephalin-like immunoreactivity to dopamine histofluorescence supports previous data demonstrating biochemical and physiological links between these two systems.     

The genus Neilo in New Zealand (Mollusca: Bivalvia)

Abstract

The phylogenetic history, paleontological implications, and ecology of larger New Zealand Recent species of Neilo (sensu stricto) are discussed. N. wairoana delli, N. blacki, and N. (Pseudomalletia) aoteana are described as new. The following taxa are reduced to chronosubspecies: N. jugifera Marwick - of N. australis (Quoy & Gaimard); N. annectens Powell and N. rugata Dell - of N. sublaevis Marwick; N. sinangula Finlay - of N. awamoana Finlay.