Mussel Hemocytes Research Articles

Transcriptomic Analysis Reveals the Wound Healing Activity of Mussel Myticin C.

Myticin C is the most studied antimicrobial peptide in the marine mussel Mytilus galloprovincialis. Although it is constitutively expressed in mussel hemocytes and displays antibacterial, antiviral, and chemotactic functions, recent work has suggested that this molecule is mainly activated after tissue injury. Therefore, the main objective of this work was to characterize the hemocytes’ transcriptomic response after a myticin C treatment, in order to understand the molecular changes induced by this cytokine-like molecule. The transcriptome analysis revealed the modulation of genes related to cellular movement, such as myosin, transgelin, and calponin-like proteins, in agreement with results of functional assays, where an implication of myticin C in the in vitro activation of hemocytes and migration was evidenced. This was also observed in vivo after a tissue injury, when hemocytes, with high concentrations of myticin C, migrated to the damaged area to heal the wound. All these properties allowed us to think about the biotechnological application of these molecules as wound healers. Human keratinocytes and larvae zebrafish models were used to confirm this hypothesis. Accelerated regeneration after a wound or tail fin amputation was observed after treatment with the myticin C peptide, supporting the chemotactic and healing activity of myticin C.

A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes

Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Trophic transfer affects cytogenetic and antioxidant responses of the mussel Mytilus galloprovincialis to copper and benzo(α)pyrene

The impacts of environmental pollutants on marine organisms can be determined by the routes of exposure. Various routes of exposure, including dietary exposure and waterborne exposure with or without feeding, were applied to study the cytogenetic responses in marine mussels Mytilus galloprovincials to typical pollutants, BaP (53.74 ± 19.79 μg/L) and Cu (47.38 ± 3.10 μg/L). The increased DNA strand breaks and micronucleus formation were found in haemocytes of mussels via the dietary exposure, indicating the vital role of trophic transfer in toxicity induction. The deeper exploration to relate BaP induced cytogenetic alterations with key antioxidant defense factors, SOD and GST, was performed under different exposure routes. The results revealed the significantly inhibited SOD activity via the trophic transfer, suggesting more direct or prompt role of SOD in antioxidant defense. On contrary, gene expressions of both sod and gst were up-regulated upon all routes of exposures, and showed negative correlation with enzyme activities. The results suggested the asynchronous regulation of studied antioxidant factors at transcriptional and enzyme functional level in mussels upon the change of exposure routes. The study brings out the first observation of trophic transfer influenced cytogenetic and antioxidant responses to pollutants and their alterative risk to marine organisms.

Characterisation of coastal aquaculture sites in the state of Rio de Janeiro, Brazil, using genotoxicity biomarkers

Mollusc aquaculture is highly dependent on environmental quality which, when impacted, can compromise cultivation. Genotoxicity biomarkers were applied to determine organism quality and environmental DNA damage capacity. The purpose of this study was to evaluate three coastal aquaculture areas with different contamination levels situated in the state of Rio de Janeiro, Brazil, where Perna perna mussels are cultivated for commercialisation and consumption. Mussel haemocytes were evaluated by genotoxic biomarkers at the DNA and chromosome levels. Organism sensitivity was tested after exposure to the carcinogen methyl methanesulfonate, which induced genetic damage when compared to the control organisms. Mussels sampled in aquaculture sites from the polluted Guanabara Bay showed the highest level of genetic damage compared to tourist areas with low urbanisation, which showed values close to zero. It can be concluded that genotoxic biomarkers, such as percentage of DNA in the comet (nucleoid) tail, obtained by the comet assay and micronucleus frequency are useful tools for recognising healthy sites for bivalve culture.

Immune Tolerance in Mytilus galloprovincialis Hemocytes After Repeated Contact With Vibrio splendidus.

Mediterranean mussels (Mytilus galloprovincialis) are sessile filter feeders that live in close contact with numerous marine microorganisms. As is the case in all invertebrates, mussels lack an adaptive immune system, but they respond to pathogens, injuries or environmental stress in a very efficient manner. However, it is not known if they are able to modify their immune response when they reencounter the same pathogen. In this work, we studied the transcriptomic response of mussel hemocytes before and after two consecutive sublethal challenges with Vibrio splendidus. The first exposure significantly regulated genes related to inflammation, migration and response to bacteria. However, after the second exposure, the differentially expressed genes were related to the control and inhibition of ROS production and the resolution of the inflammatory response. Our results also show that the second injection with V. splendidus led to changes at the transcriptional (control of the expression of pro-inflammatory transcripts), cellular (shift in the hemocyte population distribution), and functional levels (inhibition of ROS production). These results suggest that a modified immune response after the second challenge allowed the mussels to tolerate rather than fight the infection, which minimized tissue damage.

Impacts of in vivo and in vitro exposures to tamoxifen: Comparative effects on human cells and marine organisms

Tamoxifen (TAM) is a first generation-SERM administered for hormone receptor-positive (HER+) breast cancer in both pre- and post-menopausal patients and may undergo metabolic activation in organisms that share similar receptors and thus face comparable mechanisms of response. The present study aimed to assess whether environmental trace concentrations of TAM are bioavailable to the filter feeder M. galloprovincialis (100 ng L−1) and to the deposit feeder N. diversicolor (0.5, 10, 25 and 100 ng L−1) after 14 days of exposure. Behavioural impairment (burrowing kinetic), neurotoxicity (AChE activity), endocrine disruption by alkali-labile phosphate (ALP) content, oxidative stress (SOD, CAT, GPXs activities), biotransformation (GST activity), oxidative damage (LPO) and genotoxicity (DNA damage) were assessed. Moreover, this study also pertained to compare TAM cytotoxicity effects to mussels and targeted human (i.e. immortalized retinal pigment epithelium – RPE; and human transformed endothelial cells – HeLa) cell lines, in a range of concentrations from 0.5 ng L−1 to 50 μg L−1. In polychaetes N. diversicolor, TAM exerted remarkable oxidative stress and damage at the lowest concentration (0.5 ng L−1), whereas significant genotoxicity was reported at the highest exposure level (100 ng L−1). In mussels M. galloprovincialis, 100 ng L−1 TAM caused endocrine disruption in males, neurotoxicity, and an induction in GST activity and LPO byproducts in gills, corroborating in genotoxicity over the exposure days. Although cytotoxicity assays conducted with mussel haemocytes following in vivo exposure was not effective, in vitro exposure showed to be a feasible alternative, with comparable sensitivity to human cell line (HeLa).

Differential in vivo hemocyte responses to nano titanium dioxide in mussels: Effects of particle size

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in various products and inevitably released with different sizes and forms into aquatic environment. The purpose of this study was to assess the differential immune toxicity of TiO2 NPs with size difference on mussel hemocytes using flow cytometry (FCM) assays. Hemocyte parameters, including total hemocyte count (THC), hemocyte mortality (HM), phagocytosis activity (PA), lysosomal content (LC), esterase activity (EA), mitochondrial number (MN), mitochondrial membrane potential (MMP) and reactive oxygen species content (ROS) were evaluated in the mussels Mytilus coruscus exposed to two types of TiO2 NPs (25nm & 100nm: 0.1, 1, 10 mg/L, respectively). In general, size- and concentration-dependent toxicity was pronounced with 25nm-NP and highest concentration (10mg/L) being the most toxic. Alhough a slight recovery from the TiO2 exposure was observed, significant carry-over effects were still detected. These results highlight the importance of differential size effects of metal oxide NPs on toxicity mechanisms in aquatic animals.

In vitro study of apoptosis in mussel (Perna canaliculus) haemocytes induced by lipopolysaccharide

Apoptosis or programmed cell death is a fundamental process essential for an organism's development and homeostasis in the immune system of both vertebrates and invertebrates. However, little is known about apoptotic processes in marine bivalves which require further investigation to elucidate mechanisms and identify molecular effectors of cell death pathways. In this study, we characterized cellular and molecular mechanisms of apoptosis induced by lipopolysaccharide (LPS) in New Zealand Greenshell™ mussel (Perna canaliculus) haemocytes. Mussel haemocyte samples were exposed to different LPS concentrations (0, 50 and 100 μg ml−1) and incubated at 19 °C for 3 h prior to assessment of various cell health parameters via flow cytometry assays and GC/MS-based metabolomic analyses. Flow cytometry results showed slightly higher, but non-significant differences in production of reactive oxygen species between LPS-exposed and control (no LPS) samples. However, percentages of apoptotic cells determined via depolarization of the mitochondrial membrane potential and caspase-3/7 activity in LPS-exposed samples were significantly higher than in control samples, providing mechanistic information regarding initiation and progression of the apoptotic cascade. The metabolite profile of LPS-exposed haemocytes showed elevated levels of 11 metabolites compared to that of the control. These metabolites may be involved in protein and lipid degradation as a consequence of apoptosis and other immune or physiological responses. This study demonstrates that LPS could trigger apoptosis in mussel haemocytes and provides insights into apoptotic processes in mussel haemocytes. Such knowledge could be useful for understanding the immune responses of farmed bivalves to waterborne pathogens and identification of molecular biomarkers for disease management in aquaculture.

Cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes caused by environmental contamination

Environmental contamination includes a mixture of organic substances that can have detrimental effects on marine organisms and should be evaluated in the quality and risk assessment of investigated marine areas. Marine areas selected for this study are a protected area, a mariculture area, a shipyard and an industrial area. Based on the toxicity of the organic seawater extracts these areas were classified as an undisturbed reference area (S1), an area with the low anthropogenic impact (S2), a potentially endangered area (S3) and an area with high anthropogenic impact (S4) respectively. The organic mixtures present in seawater samples collected at the above defined areas were tested for the induction of DNA damage and cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes. Flow cytometric analyses were performed to detect changes in hemocytes DNA content distribution throughout the cell cycle. Organic seawater extracts from sampling sites S2, S3 and S4 induced an increase in the coefficient of variation of the G0/G1 peak and an increase in the number of cells in the G2/M phase reflecting the extent of DNA damage and G2/M arrest, respectively. The G2/M arrest in mussel hemocytes was concentration-dependent upon injection with organic seawater extracts from the S3 site and time dependant for S2, S3 and S4 sampling sites. The time dependence of the induction of the G/M arrest showed a characteristic pattern for each site due to the different quantitative and qualitative composition of the organic seawater extracts. The G2/M arrest was reversible 24 or 72 hours after treatment with organic seawater extracts from S2 or S3, and S4 sites, respectively. This reversibility was time- and site-specific indicating that such DNA damage is repairable to a certain degree according to the organic seawater extract composition. Thus, the hemocytes cell cycle alterations in the mussel Mytilus galloprovincialis caused by organic seawater extracts reliably reflect the extent of organic contamination effects for selected marine areas.

First evidence of cytotoxic effects of human protozoan parasites on zebra mussel (Dreissena polymorpha) haemocytes.

The interaction between human protozoan parasites and the immune cells of bivalves, that can accumulate them, is poorly described. The purpose of this study is to consider the mechanisms of action of some of these protozoa on zebra mussel haemocytes, by evaluating their cytotoxic potential. Haemocytes were exposed to Toxoplasma gondii, Giardia duodenalis or Cryptosporidium parvum (oo)cysts. The results showed a cytotoxic potency of the two largest protozoa on haemocytes and suggested the formation of haemocyte aggregates. Thus, this study reveals the first signs of a haemocyte:protozoan interaction.

Investigating a transcriptomic approach on marine mussel hemocytes exposed to carbon nanofibers: An in vitro/in vivo comparison

Manufactured nanomaterials are an ideal test case of the precautionary principle due to their novelty and potential environmental release. In the context of regulation, it is difficult to implement for manufactured nanomaterials as current testing paradigms identify risk late into the production process, slowing down innovation and increasing costs. One proposed concept, namely safe(r)-by-design, is to incorporate risk and hazard assessment into the design process of novel manufactured nanomaterials by identifying risks early. When investigating the manufacturing process for nanomaterials, differences between products will be very similar along key physicochemical properties and biological endpoints at the individual level may not be sensitive enough to detect differences whereas lower levels of biological organization may be able to detect these variations. In this sense, the present study used a transcriptomic approach on Mytilus edulis hemocytes following an in vitro and in vivo exposure to three carbon nanofibers created using different production methods. Integrative modeling was used to identify if gene expression could be in linked to physicochemical features. The results suggested that gene expression was more strongly associated with the carbon structure of the nanofibers than chemical purity. With respect to the in vitro/in vivo relationship, results suggested an inverse relationship in how the physicochemical impact gene expression.

Bioaccumulation of Heavy Metals and their Genotoxic Effect on Freshwater Mussel.

Contamination of fresh water bodies like riverine system is utmost concerned environmental issue. This study was aimed to assess the concentration of heavy metals in River Kabul and their bioaccumulation by freshwater mussel. Comet assay was used to evaluate the genotoxic effect of heavy metals on hemocytes of freshwater mussel. The concentration of heavy metals in water was in the order of Zn > Pb > Ni > Cu > Mn > Fe > Cr > Cd, in sediments were Fe > Zn > Cr > Ni > Mn > Pb > Cu > Cd and in the soft tissues of freshwater mussel were in order of Fe > Zn > Mn > Pb > Cu > Cr > Ni > Cd. The hemocytes of mussels from polluted sites showed significantly higher (p < 0.05) DNA damage as compared to reference site. The study showed that pollutants from industries, municipal, domestic and agricultural sources cause heavy metals contamination in River Kabul.

Responses of Mytilus galloprovincialis to challenge with the emerging marine pathogen Vibrio coralliilyticus

Vibrio coralliilyticus has emerged as a coral pathogen of concern throughout the Indo-Pacific reef. The interest towards understanding its ecology and pathogenic potential has increased since V. coralliilyticus was shown to be strongly virulent also for other species; in particular, it represents a serious threat for bivalve aquaculture, being one of the most important emerging pathogen responsible for oyster larval mortalities worldwide. V. coralliilyticus has a tightly regulated temperature-dependent virulence and it has been related to mass mortalities events of benthic invertebrates also in the temperate northwestern Mediterranean Sea. However, no data are available on the effects of V. coralliilyticus in the mussel Mytilus galloprovincialis, the most abundant aquacultured species in this area. In this work, responses of M. galloprovincialis to challenge with V. coralliilyticus (ATCC BAA-450) were investigated. In vitro, short term responses of mussel hemocytes were evaluated in terms of lysosomal membrane stability, bactericidal activity, lysozyme release, ROS and NO production, and ultrastructural changes, evaluated by TEM. In vivo, hemolymph parameters were measured in mussels challenged with V. coralliilyticus at 24h p.i. Moreover, the effects of V. coralliilyticus on mussel early embryo development (at 48 hpf) were evaluated. The results show that both in vitro and in vivo, mussels were unable to activate immune response towards V. coralliilyticus, and that challenge mainly induced lysosomal stress in the hemocytes. Moreover, V. coralliilyticus showed a strong and concentration-dependent embryotoxicity. Overall, the results indicate that, although M. galloprovincialis is considered a resistant species to vibrio infections, the emerging pathogen V. coralliilyticus can represent a potential threat to mussel aquaculture.

Using rapid assessment of marine pollution (RAMP) techniques to assess the quality of marine sediments

Sediments represent an important environmental compartment, because they provide substrate for a range of species and may accumulate contaminants in high concentrations. However, the universe of methods to assess the quality of sediments is still small. This investigation aimed to assess the quality of sediments from some sites of Ubatuba (SE Brazil). To achieve that, sediments were analyzed for the presence of PAHs, by immunoassay ELISA kit for the carcinogenic PAH (c-PAH) RaPID Assay, and for toxicity to amphipods, sea-urchin embryos and direct exposure of mussel hemocytes and measurements of the nuetral red retention time (NRTT). Results showed higher levels of PAHs in sediments from Itagua and Ribeira, those more intensely affected by contamination sources. The ecotoxicological analyses indicated these two sites as more degraded, together with the sediments from Lamberto Beach, which is also influenced by nautical activities. The NRRT correlated with the quantities of PAHs in sediments. We concluded that Ribeira Bay and Itagua Beach need more detailed investigation on pollution and that the NRRT assay exposing directly hemocytes to sediment elutriates can be a useful tool to assessing sediment quality.

Omim][BF4]-mediated toxicity in mussel hemocytes includes its interaction with cellular membrane proteins

The current study is based on the increasing demand for the assessment of ionic liquid (IL)-mediated aquatic toxicity. Specifically, although a lot of studies have been performed so far, investigating IL-mediated adverse effects on numerous aquatic organisms, little is known about their mode of action. Given that the use of in vitro models is considered as a reliable tool for determining the mediated biological effects, the modulation of specific biochemical pathways and the onset of various forms of damage with great precision and reproducibility, mixed primary cultures of mussel Mytilus galloprovincialis hemocytes were used for investigating whether 1-octyl-3-methylimidazolium tetrafluoroborate ([omim][BF4]) mediated toxicity is related to its interaction with cellular membrane proteins. Specifically, [omim][BF4]-mediated cytotoxic, oxidative and genotoxic effects were investigated in mussel hemocytes before and after pre-treatment of cells with non-toxic concentration of guanidine hydrochloride (1 mM GndHCl). The results showed that [omim][BF4] at concentrations ranging from 0.7 to 1.75 μM can induce cytotoxic (almost <50% reduction of cell viability), oxidative (increased levels of O2•− production and lipid peroxidation by-products) and genotoxic (increased levels of DNA damage) effects, while cells pre-treated with 1 mM GndHCl showed a significant attenuation of IL’s toxic potency in all cases. According to the latter, the current study showed that [omim][BF4]-mediated toxicity could be related not only to its well-known interaction with membrane lipid bilayers, but also to its interference with membrane proteins. Using GndHCl, a chaotropic agent that disrupts the hydrogen bonding network and the stability of membrane proteins via its interference with the intramolecular interactions mediated by non-covalent forces on cellular membranes, it was firstly shown that altering the membrane integrity as well as the native state of cellular membrane proteins, by weakening the hydrophobic effect, could attenuate the possible interaction of [omim][BF4] with cellular membranes and the concomitant induction of protein-based intracellular processes, commonly linked with the induction of severe cellular damage.

Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL−1), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL−1). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality.

Biological effects of tris (1-chloro-2-propyl) phosphate (TCPP) on immunity in mussel Mytilus galloprovincialis

Organophosphate flame retardants (OPFRs) are increasingly produced and used as alternatives of brominated flame-retardants (BFRs) and have become emerging marine environmental contaminants. So far, however, little is known regarding the biological effects of OPFRs in marine organisms. In this study, the biological effects of one of the most abundant OPFRs, tris (1-chloro-2-propyl) phosphate (TCPP), on the immunity in mussel Mytilus galloprovincialis were characterized by testing the reactive oxygen species, apoptosis, antioxidant system and immunity related gene expressions. Results indicated that both TCPP exposures (10 and 100 nmol L−1) significantly (p < 0.01) enhanced reactive oxygen species production and the high dose of TCPP induced more apoptosis and oxidative stress in mussel hemocytes. TCPP also induced an obvious hormesis phenomenon (low dose inhibition and high dose stimulation) in mussel hemocytes, as indicated by the gene expression profiles of caspase 8 and mytimacin. The down-regulated gene expression levels of lysozymes suggested that both TCPP exposures inhibited the innate immunity in mussel M. galloprovincialis. The significantly (p < 0.01) increased gene expression levels of TLR, galectin, PGRP and LITAF demonstrated that TCPP induced dose-dependent immune stress in mussels. Overall, this work suggested that TCPP could influence the immune system in marine mussel M. galloprovincialis.

Short-Term Exposure of Mytilus coruscus to Decreased pH and Salinity Change Impacts Immune Parameters of Their Haemocytes.

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.

Revealing Mytilus galloprovincialis transcriptomic profiles during ontogeny

Mediterranean mussels are a worldwide spread bivalve species with extraordinary biological success. One of the reasons of this success could be the reproduction strategy of bivalves, characterized by the presence of trochophore larvae. Larval development in bivalves has been a topic of raising interest in the scientific community but it deserves much more attention. The principal objective of this work was to study the transcriptomic profile of the ontogeny of Mytilus galloprovincialis analyzing the gene expression in different developmental stages, from oocytes to juveniles. For this purpose, after conducting a 454 sequencing of the transcriptomes of mussel hemocytes, adult tissues and larvae, a new DNA microarray was designed and developed.The studied developmental stages: unfertilized oocytes, veliger, pediveliger, settled larvae and juveniles, showed very different transcriptomic profiles and clustered in groups defining their characteristic gene expression along ontogeny. Our results show that oocytes present a distinct and characteristic transcriptome. After metamorphosis, both settled larvae and juveniles showed a very similar transcriptome, with no enriched GO terms found between these two stages. This suggests: 1.- the progressive loss of RNA of maternal origin through larval development and 2.- the stabilization of the gene expression after settlement.On the other hand during metamorphosis a specific profile of differentially expressed genes was found. These genes were related to processes such as differentiation and biosynthesis. Processes related to the immune response were strongly down regulated. These suggest a development commitment at the expense of other non-essential functions, which are temporary set aside. Immune genes such as antimicrobial peptides suffer a decreased expression during metamorphosis. In fact, we found that the oocytes which express a higher quantity of genes such as myticins are more likely to reach success of the offspring, compared to oocytes poor in such mRNAs, whose progeny died before reaching metamorphosis.