The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution

Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.     

Distribution of 5HT-immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary Serotonergic cell bodies and fibers were identified in the pedal ganglia of Mytilus gattoprovincialis with a serum raised against serotonin and the unlabelled peroxidase-antiperoxidase pre- and post-embedding methods. Examination of Vibratome and serial semithin sections showed that most reactive perikarya are located in the ganglionic cortex, being mainly concentrated at the medial aspect of the postero-dorsal portion of the ganglia. Immunoreactive fibers form a dense network in the neuropil, extend throughout the commissure and run parallel in the nerves and connective tracts. The morphology of serotonin-positive cells compared with that of Golgi-impregnated neurons allows the identification of a main population of unipolar, probably projecting neurons and of smaller multipolar cells likely representing local circuit elements. The ultrastructure of labelled neurons is comparable to that of serotonergic cells described in both vertebrate and invertebrate nervous systems.Supported by Ministero Pubblica Istruzione (40%)     

The HOX Gene Cluster in the Bivalve Mollusc Mytilus galloprovincialis

The clustered Hox genes play a central role in the regulation of development in bilaterian animals. In this study, we analyzed the homeobox-containing genes in a bivalve mollusc, the mussel Mytilus galloprovincialis, an unsegmented spiralian lophotrochozoan. We isolated and characterized four Hox cluster genes using the polymerase chain reaction with specific primers. Molecular alignments and phylogenetic analysis indicate that these mussel genes are homologs of the anterior group (pb ortholog), paralog group 3, and central group (PG4/Dfd and PG5/Scr) genes. The putative homeodomain sequences were designated Mgox1, Mgox2, Mgox3, and Mgox4.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.     

Glutamic acid decarboxylase (GAD)-like immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary A substance immunologically related to vertebrate glutamic acid decarboxylase (GAD) has been visualized in the pedal ganglion of Mytilus with the pre-embedding peroxidase-antiperoxidase method, by use of an antiserum raised in sheep against rat brain GAD. The results show that GAD-like immunoreactivity is present both in neuronal perikarya and in nerve fibers. Positive neurons are located mainly among the fibers of the ganglion neuropil at the commissural level, and more rarely close to unreactive cortical cell bodies. Immunoreactive nerve fibers are observed throughout the neuropil and also in cerebropedal and pedal nerves.Supported by Ministero Pubblica Istruzione (40%)     

Changes in fatty acid composition of Mytilus galloprovincialis (Lmk) fed on microalgal and wheat germ diets

Dietary fatty acid incorporation and changes in various lipid and phospholipid classes in the mussel Mytilus galloprovincialis subjected to three different dietary regimens were analysed and compared. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii, exhibiting the typical fatty acid composition of diatoms, and group C received a diet consisting of 100% wheat germ conferring a 18:2:n-6 abundance. Biochemical analyses of diets and mussels were carried out at the beginning and at the end of the 30-day experimental period. Starvation and T. weissflogii based diet poorly affected mussel growth and fatty acid composition which remained unchanged. On the contrary, the wheat germ-based diet increased the condition index and deeply affected the fatty acid profile of all lipid and phospholipid classes. The high dietary 18:2n-6 level drastically reduced tissue content of 20:4n-6, 20:5n-3 and 22:6n-3. The biosynthesis of Non Methylene Interrupted (NMI) dienoic fatty acid appeared to be insensitive to the high input of 16:1n-7 and 18:1n-9 respectively from diet B and C, and to the PUFA shortage of diet C. Nevertheless the two NMI trienoic derivatives, 20:3Δ5,11,14 and 22:3Δ7,13 16, were found higher in C with respect to other groups, presumably due to the high 18:2n-6 content of this diet.     

Effect of cobalt ions on the metabolism of some volatile and polar compounds in the marine invertebrates Mytilus galloprovincialis and Actinia equina

The compositions of the volatile and polar fractions from two coexisting Black Sea invertebrates, the mussel Mytilus galloprovincialis and the beadlet anemone Actinia equina, were established. The main metabolites in the volatile fraction from the investigated animals appeared to be methyl esters of fatty acids and fatty aldehydes. In the polar fraction from both animals low concentrations of free acids and nitrogen-containing compounds were obtained. Free carbohydrates were in much higher concentrations in M. galloprovincialis than in A. equina. Some sterols, probably as polar conjugates, were identified mainly in A. equina. Significant changes among all compounds appeared after treatment of both invertebrates with two different concentrations of cobalt ions. The variety of changes in each invertebrate could be due to their different evolutionary status. The effect of cobalt ions was often stronger at medium cobalt-ion concentrations.     

Immunocytochemical demonstration of neurotransmitters in the nerve plexuses of the foot and the anterior byssus retractor muscle of the mussel,Mytilus galloprovincialis

Summary Immunocytochemical methods were applied to study the distribution of putative neurotransmitters (5-HT, substance P, GABA, glutamate and aspartate) in the nerve plexuses of the foot and the anterior byssus retractor muscle (ABRM) of Mytilus galloprovincialis (Mollusca, Bivalvia). The foot presents extensive nerve plexuses containing 5-HT and substance P-like immunoreactive material with a similar distribution beneath the surface epithelium, around the vessels and in the glandular regions. Coexistence of the two putative neurotransmitters was observed in a few nerve fibers, Conversely, muscle fibers, both in the foot and in the ABRM, are innervated only by 5-HT-positive fibers, while substance P-like material is present only in the networks of the ABRM epimysial sheath. Immunoreactivity for glutamate and aspartate was not demonstrated, while rare GABA-positive nerve cells and fibers were found only in the foot. The results of this investigation provide a morphological background to previous physiological studies on 5-HT in the nervous system of bivalve molluscs. Moreover, they confirm that the nervous system of Mytilus contains a remarkable amount of a substance related to the vertebrate tachykinin family.     

State of the antioxidant enzyme complex in tissues of the Black Sea mollusc <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> under natural oxidative stress

The state of the antioxidant (AO) system (AOS) and the content of products of lipid peroxidation (LPO) in hepatopancreas, gill, and foot of the Black Sea mollusc Mytilus galloprovincialis Lam. were studied in norm and under conditions of natural oxidative stress (spawning). It has been established that under conditions of spawning, the AO-protection of hepatopancreas cells is predominantly oriented to inactivation of high concentrations of peroxide compounds with participation of glutathione peroxidase and catalase. In the foot tissue, on the contrary, the main role in AO-processes is played by enzymatic inhibition of superoxide anion radical with aid of superoxide dismutase. The highest level of oxidative stress in the spawning state is revealed in the mollusc gill, which is indicated by the maximal increase of the content of TBA (thiobarbituric acid)-active products.