Phylogenetic Analysis of Invertebrate Lysozymes and the Evolution of Lysozyme Function

We isolated and sequenced the cDNAs coding for lysozymes of six bivalve species. Alignment and phylogenetic analysis showed that, together with recently described bivalve lysozymes, the leech destabilase, and a number of putative proteins from extensive genomic and cDNA analyses, they belong to the invertebrate type of lysozymes (i type), first described by Jollès and Jollès (1975). We determined the genomic structure of the gene encoding the lysozyme of Mytilus edulis, the common mussel. We provide evidence that the central exon of this gene is homologous to the second exon of the chicken lysozyme gene, belonging to the c type. We propose that the origin of this domain can be traced back in evolution to the origin of bilaterian animals. Phylogenetic analysis suggests that i-type proteins form a monophyletic family. Received: 21 May 2001 / Accepted: 22 October 2001     

Cadmium binding and zinc variations in Mytilus galloprovincialis

The accumulation of Cadmium in Mytilus galloprovincialis during a 18 days exposure period was studied. Cadmium content of the mantle, viscera, gills increased throughout the time. The study of the distribution of Cd in homogenates of mussels chronically intoxicated has shown that the metal is principally bound to low mol. wt. proteins similar to metallothioneins. Mytilus galloprovincialis metallothioneins are also comparable to the vertebrate proteins, suggesting a biological function ubiquitous in the living world.     

Two forms of alpha-amylase in mantle tissue of Mytilus galloprovincialis: purification and molecular properties of form II

alpha-Amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. alpha-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis alpha-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 degrees C.     

Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels

A biomonitoring programme of wild and caged mussels (Mytilus galloprovincialis) was carried out at four selected sites along the Ligurian coast: Cornigliano, Voltri, Zinola, and Sanremo (Italy). Mussels of a very narrow size range were left in situ for 30 days. Adult specimen of mussels from natural substrates were collected in the same areas. Animals from a mussel farm located in La Spezia were used as controls. Micronucleus frequency and DNA single strand breaks, evaluated by alkaline elution, were used as biomarkers of genotoxicity. Mussels were also analyzed for polycyclic aromatic hydrocarbons (PAH) and heavy metals (Hg and Cd). Different gradients of PAH and metal concentrations were detected in tissues of mussels from different samplings sites. A weak correlation was found between single strand breaks and PAH content while MN frequency correlated with Hg concentration (r = 0.28, P < 0.002). A clear distinction between the sites, allowing classification along a pollution gradient (Sanremo < Zinola < Voltri < Cornigliano) was demonstrated by the analysis of genotoxicity parameters. The obtained results suggested that the micronucleus assay compared with DNA damage determination by alkaline elution allow to better discriminate the selected sites. DNA damage expressed as constant of elution (k ml(-1) x 10(3)) ranges from 30 +/- 9.6 to 89.60 +/- 40.10, and micronuclei frequency from 1.78 +/- 1.04 to 24.4 +/- 12.9, in control animals and in mussels from the most polluted site, respectively. Wild mussels accumulated significant concentrations of chemicals and showed a higher induction of chromosomal damage than caged mussels, expressed as micronuclei frequency. Caged mussels showed higher level of DNA damage than wild mussels, probably as a result of recent exposure. DNA damage was higher in September than in May, as opposed to micronuclei frequency being higher in May than in September. Endogenous and exogenous factors, such as change of pollutant input levels or compositions, could be considered the cause of such variability.     

Stress and immune response in the mussel Mytilus galloprovincialis

The present study investigates the effects on immune-related parameters of various stress factors (air exposure, mechanical stress, high temperature and extreme salinity conditions) faced by the bivalve mollusc Mytilus galloprovincialis during marketing procedures. We observed that some stress typologies increase phagocytosis and the number of circulating immunocytes, while others can modify immunocyte response towards a further perturbation, i.e. the marine algal toxin yessotoxin. Our results suggest that non-lethal stress can be counteracted for sometime by increasing the level of some defence parameters. Moreover, our data indicate that fishing and transport procedures could interfere with mussel immunosurveillance.     

Cadmium induction of metallothionein synthesis in Mytilus galloprovincialis

1. The accumulation and subcellular distribution of cadmium and resulting induction of metallothionein has been studied in mussels, Mytilus galloprovincialis, exposed to 400 μg Cd 1⁻¹.2. Cadmium accumulated by mussels was primarily associated with metallothionein: a maximum four-fold increase in concentrations of the metal-binding protein (3 mg g⁻¹ to 12 mg g⁻¹) was observed after 30 days.3. Results for M. galloprovincialis confirm the importance of metallothionein in the detoxification of intracellular cadmium and indicate that induction rates are identical to those observed in the closely-related M. edulis.4. Intercomposition of data between these species may therefore be valid when employing metallothionein assays to determine sub-lethal response to cadmium contamination.     

Cadmium accumulation in tissues of the mussel Mytilus galloprovincialis

Studies have been made on cadmium accumulation in tissues of mussels kept within 20-60 days in water artificially enriched by Cd up to 20-100 micrograms/l. Irrespectively of cadmium concentration in the medium, its accumulation in tissues decreases in the following order: mid-gut gland, gills, gonads, mantle, adductor. Maximum concentration of Cd was found in the digestive tubuli of the mid-gut gland by X-ray microanalysis. The increase in S and, to a lower extent, P concentrations in these tubuli was also observed. It is suggested that the latter is due to immobilization of Cd by metal-binding proteins as well as to lyzosomal vesicles involved into detoxication of Cd. The increase in the external cadmium up to 100 micrograms/l did not affect the level of K, Ca and Mg in tissues of the mussel.     

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.     

Optimising settlement assays of pediveligers and plantigrades of Mytilus galloprovincialis

The mussel Mytilus galloprovincialis is a common aquaculture species, and also a major fouling organism that has negative economic impacts. There are no standard assay conditions for this important species and therefore, this study quantified the effect of key factors on the settlement of pediveligers and plantigrades. Density dependent settlement did not occur for either pediveligers or plantigrades. Settlement increased in drop assays in a 12 h light:12 h dark cycle, while bottom shade had no effect of any magnitude. In addition, settlement was significantly enhanced by storing pediveligers for between 4 and 24 days at 4 °C. Overall, these data provide the template to optimise and standardise static laboratory settlement assays for mussels in order to develop materials that either enhance settlement for the aquaculture industry, or deter settlement for antifouling applications. Furthermore, simple mechanisms such as storage at 4 °C can enhance settlement beyond current methods used in aquaculture hatcheries.     

Optimising settlement assays of pediveligers and plantigrades of Mytilus galloprovincialis

The mussel Mytilus galloprovincialis is a common aquaculture species, and also a major fouling organism that has negative economic impacts. There are no standard assay conditions for this important species and therefore, this study quantified the effect of key factors on the settlement of pediveligers and plantigrades. Density dependent settlement did not occur for either pediveligers or plantigrades. Settlement increased in drop assays in a 12 h light:12 h dark cycle, while bottom shade had no effect of any magnitude. In addition, settlement was significantly enhanced by storing pediveligers for between 4 and 24 days at 4°C. Overall, these data provide the template to optimise and standardise static laboratory settlement assays for mussels in order to develop materials that either enhance settlement for the aquaculture industry, or deter settlement for antifouling applications. Furthermore, simple mechanisms such as storage at 4°C can enhance settlement beyond current methods used in aquaculture hatcheries.     

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.     

Adaptive Evolution of Defense Ability Leads to Diversification of Prey Species

In this paper, by using the adaptive dynamics approach, we investigate how the adaptive evolution of defense ability promotes the diversity of prey species in an initial one-prey–two-predator community. We assume that the prey species can evolve to a safer strategy such that it can reduce the predation risk, but a prey with a high defense ability for one predator may have a low defense ability for the other and vice versa. First, by using the method of critical function analysis, we find that if the trade-off is convex in the vicinity of the evolutionarily singular strategy, then this singular strategy is a continuously stable strategy. However, if the trade-off is weakly concave near the singular strategy and the competition between the two predators is relatively weak, then the singular strategy may be an evolutionary branching point. Second, we find that after the branching has occurred in the prey strategy, if the trade-off curve is globally concave, then the prey species might eventually evolve into two specialists, each caught by only one predator species. However, if the trade-off curve is convex–concave–convex, the prey species might eventually branch into two partial specialists, each being caught by both of the two predators and they can stably coexist on the much longer evolutionary timescale.     

Two events are responsible for an insertion in a paternally inherited mitochondrial genome of the mussel Mytilus galloprovincialis

Frequent nonhomologous recombination has been previously postulated to explain the 1045-bp insertion in one mitochondrial sperm-transmitted haplotype of Mytilus galloprovincialis. Such recombination would lead to the disruption of gene order and so the existence of a specific mechanism for maintaining the same gene order in both mitochondrial genomes of Mytilus has been proposed. Here the simpler explanation of the observed structure, involving a tandem duplication and a deletion, is presented. Their occasional occurrence in Mytilus mtDNA proves the similarity, not the difference, between animals with and without DUI.