Effect of cobalt ions on lipid and sterol metabolism in the marine invertebrates Mytilus galloprovincialis and Actinia equina

The lipid and sterol fractions of the mussel Mytilus galloprovincialis and the anemone Actinia equina were analyzed before and after incubation with cobalt ions. There were significant changes in the lipid and sterol composition, strongly depending on the cobalt ions concentrations. The changes in the lipid composition of both invertebrates were different for neutral lipids and phospholipids. These changes concern the length of the fatty acid chains as well as their unsaturation. The cobalt ions inhibited the oxidation of sterols and their alkylation at C-24. Because of these results we can assume that some of the changes caused by cobalt ions might possess adaptive value.     

An RFLP assay to determine if Mytilus galloprovincialis Lmk. (Mytilidae; Bivalvia) is of Northern or Southern hemisphere origin

Mytilus galloprovincialis is one of three smooth shelled blue mussel species belonging to the Mytilus edulis species complex. Naturally occurring and introduced populations of M. galloprovincialis are widely distributed throughout many regions of the globe. Mytilus galloprovincialis includes morphologically indistinguishable Northern and Southern hemisphere mtDNA lineages that have been separated for ～1 my. To distinguish recently introduced Northern M. galloprovincialis from resident Southern M. galloprovincialis in New Zealand, we developed a 16s rRNA RFLP assay. We compared RFLP assignments of 178 mussels with those generated from a 16s rRNA sequence-estimated phylogeny. All mussels were correctly assigned by the RFLP to their sequence-based phylogenetic placement. This assay allows the rapid identification of Northern and Southern hemisphere M. galloprovincialis and will provide an important tool for monitoring human mediated introductions of otherwise cryptic lineages.     

Evidence of selective mortality in favour of the Mytilus galloprovincialis Lmk phenotype in British mussel populations

Samples of mussels ( Mytilus ) were collected from 17 localities within hybrid zones of Mytilus edulis and Mytilus galloprovincialis in south-west and north-east England. The study of two polymorphic allozyme loci ( esterase-D and octopine dehydrogenase ), which are partially diagnostic for the two forms of mussel, reveal the existence of widespread length-dependent allele frequency variation. Larger mussels tend to have a higher frequency of alleles characteristically at high frequency in Mytilus galloprovincialis. Also at a given shell length galloprovincialis alleles have a higher frequency higher up the shore. Computer simulation is used to demonstrate that length-dependent variation may be generated not only by differential mortality but also by differential growth and in models including or excluding immigration. Evidence supports the hypothesis that selective mortality acting in favour of the galloprovincialis phenotype within hybrid populations in Britain is balanced by immigration of the more abundant Mytilus edulis.     

Mitochondrial contribution to metabolic changes in the digestive gland of Mytilus galloprovincialis during anaerobiosis

The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis. The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107-113, 2000.     

Nitrogen availability influences the biochemical composition and photosynthesis of tank-cultivated Ulva rigida (Chlorophyta)

Physiological and biochemical changes in relation to inorganic nitrogen availability were studied for tank-cultivated Ulva rigida grown under nitrogen- enriched and nitrogen-depleted seawater. U. rigida was initially cultivated in nitrogen-enriched seawater (daily concentrations of NH4+ and NO3- + NO2- ranged between 0.5–1.7 and 0.06–0.15 mg L-1, respectively), then transferred to nitrogen-depleted seawater where photosynthetic capacity decreased to zero after 23 d. At the time (14 d) when photosynthetic rates were lower than 2.0 μmol O2 g-1 FW min-1 and strong bleaching had occurred, some algae were returned to the initial nitrogen-enriched seawater to study recovery from N-limited growth. Data on biochemical composition (chlorophylls, ash, caloric content, fatty acids and dietary fibres) and colouration varied significantly depending on the nitrogen conditions. C:N ratios correlated significantly with biochemical parameters. Fatty acid (FA) synthesis continued during the N-starvation period; saturated and mono-unsaturated FA increased to a maximun of 72.2%, while poly-unsaturated fatty acids (PUFA) decreased to 27.7%. During the N-enriched recovery period, the reverse was found. C:N ratios above 10 correlated with carbohydrate synthesis as shown by the dietary fibre level. Under nitrogen enriched conditions, C:N ratios decreased along with a decrease in fibre level. Under controlled conditions, nitrogen represents a major influence on the development of intensive tank cultivation of Ulva rigida, not only by affecting parameters closely related to nitrogen metabolism but also some clearly influenced by carbon uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

The chitin synthase involved in marine bivalve mollusk shell formation contains a myosin domain

Chitin is a key component in mollusk nacre formation. However, the enzyme complex responsible for chitin deposition in the mollusk shell remained unknown. We cloned and characterized the chitin synthase of the marine bivalve mollusk Atrina rigida. We present here the first chitin synthase sequence from invertebrates containing an unconventional myosin motor head domain. We further show that a homologous gene for chitin synthase is expressed in the shell forming tissue of larval Mytilus galloprovincialis even in early embryonic stages. The new data presented here are the first clear-cut indication for a functional role of cytoskeletal forces in the precisely controlled mineral deposition process of mollusk shell biogenesis.     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

Electrophoretic investigation of systematic relationships in the marine mussels Modiolus modiolus L., Mytilus edulis L., and Mytilus galloprovincialis Lmk. (Mytilidae; Mollusca)

Genetic variation was assayed electrophoretically at 13–16 loci in Modiolus modiolus, Mytilus edulis, and Mytilus galloprovincialis. High genetic distance ( D ) values were observed between Modiolus modiolus and Mytilus edulis (1.516 ± 0.523) and between Modiolus modiolus and Mytilus galloprovincialis (1.564 ± 0.539), whereas the distance between Mytilus edulis and M. galloprovincialis (0.167 ± 0.118) was rather low. The systematic status ot Mytilus edulis and M. galloprovincialis is discussed in relation to these lindings and the genetic distance values are used to estimate divergence times which in turn are compared with paleontological estimates. The observations of high average heterozygosity in Modiolus modiolus, and high correlations of locus heterozygosity between taxa are discussed briefly.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

Peculiarities of the corrective effects of polar lipids and bioantioxidants from sea hydrobionts in impairments of lipid and carbohydrate metabolism

A total mixture of phospho- and glycolipids from sea macrophytes Sargassum pallidum, Ulva fenestrata, Zostera marinawas separated and the fatty acid composition was determined. Biological activity of the mixtures of polar lipids and natural antioxidants (echinochrome A from the flat sea urchin Scaphechinus mirabilis and a polyphenolic complex from the sea grass Zostera marina) were studied under conditions of impairments of carbohydrate and lipid metabolism. Doses and compositions of mixtures of polar lipids and antioxidants possessing high corrective activity were optimized in mice with the experimental model of atherosclerosis and diabetes. Based on these results possible mechanisms of the effects of polar lipids containing various polyunsaturated fatty acids and the investigated antioxidants have been proposed. The developed compositions may be used for creation of new biologically active additives and drugs.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

Identification of octopine dehydrogenase from Mytilus galloprovincialis

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M. galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate L-arginine (88.22%), compared to L-alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or β-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M. galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate (P. maximus).     

Genomic reticulation indicates mixed ancestry in Southern-Hemisphere Mytilus spp. mussels

Previous surveys of allozyme variation in smooth-shell Mytilus spp. mussels have reported the presence in the Southern Hemisphere of both Mytilus edulis and Mytilus galloprovincialis mussels. In the present study, nuclear DNA markers mac-1 and Glu-5 '/ Glu-3 ', both diagnostic for Northern-Hemisphere M. edulis and M. galloprovincialis , were used to further characterize the nuclear genomes of M. edulis from Kerguelen and M. galloprovincialis from Tasmania. Genomic reticulation was observed, with typical M. edulis allelomorphs fixed in both populations at locus mac-1 whereas, at locus Glu-5 '/ Glu-3 ', allelomorphs characteristic of M. galloprovincialis were present in Kerguelen and nearly fixed in Tasmania. Kerguelen mussels had a genome of mixed M. edulis and M. galloprovincialis ancestry without evidence of barriers to merging as shown by Hardy–Weinberg and linkage equilibrium. Tasmanian mussels possessed a predominantly M. galloprovincialis genomic background introgressed by M. edulis allelomorphs at locus mac-1 . Genetic drift superimposed on ancient hybridization and introgression may explain the genomic reticulation observed in both Kerguelen and Tasmanian mussels. There was no evidence of a recent introduction of Northern-Hemisphere M. galloprovincialis or M. edulis to Kerguelen or Tasmania.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 747–754.     

The mediterranean mussel Mytilus galloprovincialis Lmk. in Japan

Mussels ( Mytilus sp.) from Sanriku Bay, NE Honshu, Japan were examined using morphological characters and electrophoretically detectable enzyme polymorphisms. Using both sets of criteria, the mussels were identified as M. galloprovincialis , the mediterranean mussel. This confirms an earlier opinion, which was based on morphological criteria alone, that the mediterranean mussel occurs on the mainland coast of Japan. Investigation of some early Japanese literature suggests that mussels did not occur in this area earlier this century, and M. galloprovincialis may have been introduced to the region of Kobe, around 1930–1935. The present-day distribution of M. edulis and M. galloprovincialis in the Japanese archipelago may be explained by sea-surface temperatures in the region.     

Three species of Ulva (Ulvophyceae) from the Persian Gulf as potential sources of protein,essential amino acids and fatty acids

Three species of the cosmopolitan genus Ulva (U. paschima , U. chaugulii and U. ohnoi ) from the Persian Gulf were identified using morphological and molecular markers, and were analyzed for total lipids, fatty acids, proteins and amino acid profiles. Our results show that the studied Ulva species have high protein content (9?25% dry weight), contain essential fatty acids and nearly all essential amino acids. This suggests Ulva could have potential as a form of human nutrition and animal feed with a low carbon footprint.     

First record of the microsporidian parasite Steinhausia mytilovum in Mytilus sp. (Bivalvia: Mytilidae) from France

Steinhausia mytilovum is a globally distributed microsporidian parasite which infects the oocytes of the blue mussels Mytilus edulis and M. galloprovincialis. Despite the intensive monitoring effort made on mussel populations, the parasite has not previously been reported in France. We report herein on the occurrence of S. mytilovum in Mytilus sp. from 1 cultured and 2 natural populations on the northern coast of France, thus extending the parasite's known distribution northwards. We also report on the observation in 1989 of S. mytilovum in M. galloprovincialis from the Golfe de Fos area in the Mediterranean Sea (South of France). S. mytilovum was observed in the European hybrid zone between M. edulis and M. galloprovincialis, which therefore renders the exact taxonomic status of the infected hosts unknown. The prevalence of the parasite was low, which suggests that its effect on mussel populations was probably limited.     

Patterns of polymorphism and gene flow of gender-associated mitochondrial DNA lineages in European mussel populations

Mussels of the genus Mytilus have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally and the F type is transmitted maternally. RFLP analysis is used to assess phylogenetic relationships and nucleotide diversity and divergence for both mtDNA genomes in European populations of M. edulis and Atlantic and Mediterranean forms of M. galloprovincialis. Ten restriction endonucleases were used to assay variation in regions of the ND2 and COIII genes for a total of 77 individuals. F and M genomes show a concordant phylogenetic split into two major divergent clades, one specific to Mediterranean M. galloprovincialis and the other containing haplotypes from the three taxa. For both genomes, the geographical distribution of mtDNA variation suggests: (i) extensive levels of mtDNA introgression; (ii) asymmetric mtDNA gene flow from Atlantic to Mediterranean populations; and (iii) recurrent historical hybridization events. Significantly higher mtDNA diversity and divergence are observed for the M than F genome in all three Mytilus taxa, although the evolutionary forces responsible for these differences cannot be resolved. The extensive mtDNA gene flow between European Mytilus taxa conflicts with the restricted mtDNA introgression observed in American mussels , implying geographical variation in the nature of nuclear/mtDNA interactions regulating biparental inheritance.     

Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida

Chlorophyll synthesis is stimulated by red light in the green alga Ulva rigida C. Ag. and in the red alga Porphyra umbilicalis (L.) Kützing. Because the effect of red light showed some far-red reversibility in successive red and far-red light treatments, the involvement of phytochrome or a phytochrome-like photoreceptor is suggested. The extent of the response is dependent on exposure and photon fluence rate of red-light pulses. In addition to the effect of red light, a strong stimulation of chlorophyll synthesis by blue light was only observed in Ulva rigida. The effect of blue light shows also some far-red reversibility. In the green alga the accumulated chlorophyll is higher after blue light pulses than after red light pulses. In Porphyra umbilicalis , however, the contrary is observed. In Ulva rigida the involvement of a blue light photoreceptor in addition to phytochrome or a phytochrome-like photoreceptor is proposed. The different responses to red and blue light in both algae are explained in terms of their adaptation to the natural light environment.     

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels (Mytilus edulis x M. galloprovincialis)

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis x M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis x M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.     

Distribution of substance P-like immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Nerve cell bodies and fibers displaying Substance P-like (SP-li) immunoreactivity have been identified in the pedal ganglia (PG) of Mytilus galloprovincialis. Most reactive neurons are small-sized, unipolar and are located in the ganglionic cortex. Positive fibers form a dense network in the neuropile and run parallel in the commissure and in the nerves and connective tracts. The positivity of the labelled elements to a monoclonal antibody indicates that SP-li substance of Mytilus contains a portion immunologically similar to the C-terminal pentapeptide responsible for the pharmacological activity of vertebrate SP.