Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17 degrees C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10^?4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC₅₀) for chlorpromazine and amitriptyline was 1.6 × 10^?6 M and 6.6 × 10^?5 M, respectively. Idazoxan was less effective with an IC₅₀ of 4.4 × 10¹³ M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K⁺ was not inhibited by 10^?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Induction of metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis Lamarck, 1819 using neuroactive compounds,KCl, NH4Cl and organic solvents

Pediveliger larvae of Mytilus galloprovincialis were subjected to a series of bioassays to investigate the induction of metamorphosis using neuroactive compounds, K⁺, NH₄ ⁺ and organic solvents. Growth and survival of post-larvae obtained using ethanol and methanol were also observed. Epinephrine, phenylephrine, clonidine and metanephrine induced larval metamorphosis at 10^?6 to 10^?4 M in both 24-h and continuous exposure assays. In 24-h exposure assays, α-methyldopa at 5×10^?5 M and methoxyphenamine at 5×10^?5?10^?4 M induced 55?94% metamorphosis. Similarly, excess K⁺ at 3×10^?2 M induced 39% metamorphosis and NH₄ ⁺ at 1?5×10^?2 M induced 63–78% metamorphosis. The EC50s of seven organic solvents ranged from 0.04 to 0.82 M. Post-larvae that metamorphosed using ethanol and methanol survived as juveniles and grew at the same rate as those from microbial biofilm. Thus, the above compounds can be useful inducers of metamorphosis for antifouling studies using larvae and juveniles of M. galloprovincialis.     

The effect of chlorpyrifos on protein content and acid DNase activity in the mussel,Mytilus galloprovincialis

We evaluated the effects of short-term exposure to an organophosphate pesticide chlorpyrifos on the digestive gland and gills of the mussel Mytilus galloprovincialis. We studied metabolic activity by quantifying protein content and physiological function responses using acid DNase activity. The increase in protein content was observed in both the target tissues of mussels exposed to 0.03 μg/L chlorpyrifos when compared with control mussels. The pattern of acid DNase activity in digestive gland and gills indicated a tissue-specific response, although the lowest concentration of chlorpyrifos caused changes in acid DNase activity in both tissues. In the digestive gland, the increase of acid DNase activity was observed in mussel exposed to 0.03 μg/L chlorpyrifos, followed by decrease up to 100 μg/L chlorpyrifos. Enzyme activity in the gills showed a dose response effect. The results support the use of acid DNase activity in the digestive gland as a sensitive response to an environmentally relevant range of pesticide concentrations. It may also indicate an effect on mussel physiological status.     

Investigation of the loss of byssus in Mytilus galloprovincialis from mussel farms in the Adriatic Sea

A fungal infection has been found in the mussel Mytilus galloprovincialis from Adriatic Sea mussel farms. The infection ultimately results in the loss of the byssus, with serious consequences for mussel farming yield. The pathogen provokes the progressive destruction of the foot muscles, also damaging related structures such as the intra-organism part of the byssus apparatus, resulting in loss of the thread component. The affected health status of the animal is also sustained by modifications in the digestive gland structure, ranging from hyperactivity to extreme cell death in the tubula. At present, the identity of the harmful fungus is unknown.     

Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine‐like protein properties

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine‐like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl₂, and it was tested the PL‐proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL‐proteins of summer mussels that were produced on PL‐proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid‐urea polyacrylamide gels (AU‐PAGE) and sodium dodecyl sulfate‐PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL‐proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock‐associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.     

Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 μg Cd l^–1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered. Electronic Publication     

The impact of the sea anemone Actinothoe sphyrodeta on Mytilus galloprovincialis mussel cultivation (Galicia,Spain)

Abstract

Marine mussel aggregations act as a substratum and refuge for many fouling species. Mussel cultivation in Galicia, Spain, is carried out on hanging ropes in subtidal systems. The fauna associated with this cultivation includes a large number of invertebrates that compete for space or food with the mussels, or use their clusters as a refuge from predators or water turbulence. Outbreaks of the epibiont anemone Actinothoe sphyrodeta have been reported in cultivated Galician mussels since 2013, but their impact has not been investigated rigorously. Here, the temporal and spatial variability of Actinothoe sphyrodeta on mussel shells throughout one year is presented. Sampling of mussel size, weight and byssus attachment strength allowed mussel tenacity (attachment strength relative to size) to be calculated. A higher presence of Actinothoe sphyrodeta correlated with lower mussel tenacity and greater biomass losses, suggesting that this species could be an economically important biofouling component.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10?? M epinephrine. The concentration that inhibited metamorphosis by 50% (IC??) for chlorpromazine and amitriptyline was 1.6 x 10?? M and 6.6 x 10?? M, respectively. Idazoxan was less effective with an IC?? of 4.4 x 1013 M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K+ was not inhibited by 10?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Histopathological indices and inflammatory response in the digestive gland of the mussel Mytilus galloprovincialis as biomarker of immunotoxicity to silver nanoparticles

Background: Histopathological assessments approaches in bivalves have become an important tool in environmental toxicology. This study seeks to develop a quantitative histopathological index (Ih) and inflammation score as biomarkers in the aim to assess the health status of nanoparticles exposed mussels.

Methods: Digestive gland hematoxylin and eosin (H&;E) stained sections from Mytilus galloprovincialis were assessed after in vivo exposure (for 3, 6 and 12?h) to silver nanoparticles (Ag-NPs?Results: Silver nanoparticles clearly induced histopathological alterations in digestive gland (maximum inflammation 2.75 with AgNP?p?Ih with AgNP?p?Ih were recorded after uptake routes were blockade: AgNP?p?Conclusions: Histopathological assessments showed to be promising tool in nanotoxicity which seems to depend on nanoparticles size, exposure time and interestingly to uptake routes. It was not clear: is it the length of exposure or the size of particles is more impactful.     

Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. galloprovincialis

Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine mussels Mytilus edulis and M. galloprovincialis, using three length-polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient from M. galloprovincialis on the Iberian Peninsula to M. edulis populations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical of M. galloprovincialis first decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantly M. edulis-like and M. galloprovincialis-like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre- or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory 'short-cut' connecting M. edulis-like populations of the Charente patch to an external M. edulis population in Normandy and thought to reflect artificial transfer of spat for aquaculture.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

Assortative fertilization and selection at larval stage in the mussels Mytilus edulis and M. galloprovincialis

Assortative mating (prezygotic isolation) and reduced hybrid fitness (postzygotic isolation) are typically invoked to explain the stability of hybrid zones. In the tension zone model, these factors work in opposition to migration, which promotes genetic homogeneity. Many marine animals migrate over long distances through a planktonic larval stage. Therefore, strong reproductive isolation is needed to maintain stable marine hybrid zones. However, surprisingly little is known about mating preferences and hybrid fitness in marine organisms. Smooth-shelled mussels (Mytilus spp.) form a well-known species complex, with hybridization over extensive areas such as the contact zone of M. edulis and M. galloprovincialis around European Atlantic coasts. This paper reports direct experimental evidence of assortative fertilization, hybrid larval inviability, and early heterosis for growth rate in M. edulis and M. galloprovincialis. Four crosses between pure M. edulis and M. galloprovincialis were analyzed with a new polymerase-chain-reaction-based diagnostic marker. Gamete competition between taxa was allowed in two out of the four crosses. Genotype frequencies observed at an early stage (36 h after fertilization) unambiguously revealed assortative fertilization when gamete competition was allowed. A significant reduction in hybrid viability was subsequently observed during the larval stage. At the same stage an antagonistic effect, heterosis, was observed on growth rate. However, even if heterosis is observed in the F1, it is expected to vanish in subsequent hybrid generations. Although specialization for different habitats and asynchronous spawning have been mentioned as factors contributing to the maintenance of the blue mussel hybrid zone in Europe, we argue that assortative fertilization and reduced hybrid fitness are important factors that also contribute to the stabilization of this zone. These results emphasize that multiple factors may act concomitantly in a barrier to gene flow, especially in complex life cycles. Furthermore, they show that assortative mating through gamete preference, as already demonstrated for sea urchins, may play a role in speciation processes taking place in the sea.     

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.     

Induction of metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis Lamarck, 1819 using neuroactive compounds, KCl, NH4Cl and organic solvents

Pediveliger larvae of Mytilus galloprovincialis were subjected to a series of bioassays to investigate the induction of metamorphosis using neuroactive compounds, K(+), NH(4)(+) and organic solvents. Growth and survival of post-larvae obtained using ethanol and methanol were also observed. Epinephrine, phenylephrine, clonidine and metanephrine induced larval metamorphosis at 10(-6) to 10(-4) M in both 24-h and continuous exposure assays. In 24-h exposure assays, alpha-methyldopa at 5 x 10(-5) M and methoxyphenamine at 5 x 10(-5)-10(-4) M induced 55-94% metamorphosis. Similarly, excess K(+) at 3 x 10(-2) M induced 39% metamorphosis and NH(4)(+) at 1-5 x 10(-2) M induced 63-78% metamorphosis. The EC50s of seven organic solvents ranged from 0.04 to 0.82 M. Post-larvae that metamorphosed using ethanol and methanol survived as juveniles and grew at the same rate as those from microbial biofilm. Thus, the above compounds can be useful inducers of metamorphosis for antifouling studies using larvae and juveniles of M. galloprovincialis.     

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: a biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Phenoloxidase (E.C. 1.14.18.1) from the byssus gland of Mytilus edulis: Purification,partial characterization and application for screening products with potential antifouling activities

Although a total ban on the use of TBT coatings is not expected in the short term, there is a growing need for environmentally safe antifouling systems. To assist in the rapid screening of a large number of potential antifouling substances, a method that is simple, efficient and inexpensive is required. The production of byssus threads by the blue mussel, Mytilus edulis, has often been studied for testing the antifouling efficacy of various compounds. The present study reports a new antifouling assay based on the inhibition of purified M. edulis phenoloxidase activity. The method has the advantage of being specific, reliable, sensitive and rapid.     

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.