The Mediterranean mussel Mytilus galloprovincialis: a novel model for developmental studies in mollusks.

A model organism in developmental biology is defined by its experimental amenability and by resources created for the model system by the scientific community. For the most powerful invertebrate models, the combination of both has already yielded a thorough understanding of developmental processes. However, the number of developmental model systems is still limited, and their phylogenetic distribution heavily biased. Members of one of the largest animal lineages, the Spiralia, for example, have long been neglected. In order to remedy this shortcoming, we have produced a detailed developmental transcriptome for the bivalve mollusk Mytilus galloprovincialis, and have expanded the list of experimental protocols available for this species. Our high-quality transcriptome allowed us to identify transcriptomic signatures of developmental progression and to perform a first comparison with another bivalve mollusk: the Pacific oyster Crassostrea gigas. To allow co-labelling studies, we optimized and combined protocols for immunohistochemistry and hybridization chain reaction to create high-resolution co-expression maps of developmental genes. The resources and protocols described here represent an enormous boost for the establishment of Mytilus galloprovincialis as an alternative model system in developmental biology.

Hedgehog signaling is required for larval muscle development and larval metamorphosis of the mussel Mytilus coruscus.

Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh, McPtc, McSmo, and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates.

Expansion and diversity of caspases in Mytilus coruscus contribute to larval metamorphosis and environmental adaptation.

BACKGROUND: Apoptosis is involved (directly and indirectly) in several physiological processes including tissue remodeling during the development, the turnover of immune cells, and a defense against harmful stimuli. The disordered apoptotic process participates in the pathogenesis of various diseases, such as neoplasms, and chronic inflammatory or systemic autoimmune diseases, which are associated with its inadequate regulation. Caspases are vital components of the apoptotic pathway that are involved in developmental and immune processes. However, genome-wide identification and functional analysis of caspase have not been conducted in Mytilus coruscus, which is an economically important bivalve. RESULTS: Here, 47 caspase genes were identified from the genomes of M. coruscus, and the expansion of caspase-2/9 and caspase-3/6/7 genes were observed. Tandem duplication acts as an essential driver of gene expansion. The expanded caspase genes were highly diverse in terms of sequence, domain structure, and spatiotemporal expression profiles, suggesting their functional differentiation. The high expression of the expanded caspase genes at the pediveliger larvae stage and the result of apoptosis location in the velum suggest that the apoptosis mediated by them plays a critical role in the metamorphosis of M. coruscus larvae. In gill, caspase genes respond differently to the challenge of different strains, and most caspase-2/9 and caspase-3/6/7 genes were induced by copper stress, whereas caspase-8/10 genes were suppressed. Additionally, most caspase genes were upregulated in the mantle under ocean acidification which could weaken the biomineralization capacity of the mantle tissue. CONCLUSIONS: These results provide a comprehensive overview of the evolution and function of the caspase family and enhanced the understanding of the biological function of caspases in M. coruscus larval development and response to biotic and abiotic challenges.

Marine transmissible cancer navigates urbanized waters, threatening spillover.

Inter-individual transmission of cancer cells represents a unique form of microparasites increasingly reported in marine bivalves. In this study, we sought to understand the ecology of the propagation of Mytilus trossulus Bivalve Transmissible Neoplasia 2 (MtrBTN2), a transmissible cancer affecting four Mytilus mussel species worldwide. We investigated the prevalence of MtrBTN2 in the mosaic hybrid zone of M. edulis and M. galloprovincialis along the French Atlantic coast, sampling contrasting natural and anthropogenic habitats. We observed a similar prevalence in both species, probably due to the spatial proximity of the two species in this region. Our results showed that ports had higher prevalence of MtrBTN2, with a possible hotspot observed at a shuttle landing dock. No cancer was found in natural beds except for two sites close to the hotspot, suggesting spillover. Ports may provide favourable conditions for the transmission of MtrBTN2, such as high mussel density, stressful conditions, sheltered and confined shores or buffered temperatures. Ships may also spread the disease through biofouling. Our results suggest ports may serve as epidemiological hubs, with maritime routes providing artificial gateways for MtrBTN2 propagation. This highlights the importance of preventing biofouling on docks and ship hulls to limit the spread of marine pathogens hosted by fouling species.

SNP genotyping revealed a hybrid zone between Mytilus chilensis and M. platensis in southern South America (the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands).

Hybrid zones among mussel species have been extensively studied in the northern hemisphere. In South America, it has only recently become possible to study the natural hybrid zones, due to the clarification of the taxonomy of native mussels of the Mytilus genus. Analysing 54 SNP markers, we show the genetic species composition and admixture in the hybrid zone between M. chilensis and M. platensis in the southern end of South America. Bayesian, non-Bayesian clustering and re-assignment algorithms showed that the natural hybrid zone between M. chilensis and M. platensis in the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands shows clinal architecture. The hybrid zone can be divided into three different areas: the first one is on the Atlantic coast where only pure M. platensis and hybrid were found. In the second one, inside the Strait of Magellan, pure individuals of both species and mussels with variable degrees of hybridisation coexist. In the last area at the Strait in front of Punta Arenas City, fjords on the Isla Grande de Tierra del Fuego, and at the Beagle Channel, only M. chilensis and a low number of hybrids were found. According to the proportion of hybrids, bays with protected conditions away from strong currents would give better conditions for hybridisation. We do not find evidence of any other mussel species such as M. edulis, M. galloprovincialis, M. planulatus or M. trossulus in the zone.

Patterns of admixture and introgression in a mosaic Mytilus galloprovincialis and Mytilus edulis hybrid zone in SW England.

The study of hybrid zones offers important insights into speciation. Earlier studies on hybrid populations of the marine mussel species Mytilus edulis and Mytilus galloprovincialis in SW England provided evidence of admixture but were constrained by the limited number of molecular markers available. We use 57 ancestry-informative SNPs, most of which have been mapped genetically, to provide evidence of distinctive differences between admixed populations in SW England and asymmetrical introgression from M. edulis to M. galloprovincialis. We combine the genetic study with analysis of phenotypic traits of potential ecological and adaptive significance. We demonstrate that hybrid individuals have brown mantle edges unlike the white or purple in the parental species, suggesting allelic or non-allelic genomic interactions. We report differences in gonad development stage between the species consistent with a prezygotic barrier between the species. By incorporating results from publications dating back to 1980, we confirm the long-term stability of the hybrid zone despite higher viability of M. galloprovincialis. This stability coincides with a dramatic change in temperature of UK coastal waters and suggests that these hybrid populations might be resisting the effects of global warming. However, a single SNP locus associated with the Notch transmembrane signalling protein shows a markedly different pattern of variation to the others and might be associated with adaptation of M. galloprovincialis to colder northern temperatures.

Transgressive gene expression and expression plasticity under thermal stress in a stable hybrid zone.

Interspecific hybridization can lead to myriad outcomes, including transgressive phenotypes in which the hybrids are more fit than either parent species. Such hybrids may display important traits in the context of climate change, able to respond to novel environmental conditions not previously experienced by the parent populations. While this has been evaluated in an agricultural context, the role of transgressive hybrids under changing conditions in the wild remains largely unexplored; this is especially true regarding transgressive gene expression. Using the blue mussel species complex (genus Mytilus) as a model system, we investigated the effects of hybridization on temperature induced gene expression plasticity by comparing expression profiles in parental species and their hybrids following a 2-week thermal challenge. Hybrid expression plasticity was most often like one parent or the other (50%). However, a large fraction of genes (26%) showed transgressive expression plasticity (i.e. the change in gene expression was either greater or lesser than that of both parent species), while only 2% were intermediately plastic in hybrids. Despite their close phylogenetic relationship, there was limited overlap in the differentially expressed genes responding to temperature, indicating interspecific differences in the responses to high temperature in which responses from hybrids are distinct from both parent species. We also identified differentially expressed long non-coding RNAs (lncRNAs), which we suggest may contribute to species-specific differences in thermal tolerance. Our findings provide important insight into the impact of hybridization on gene expression under warming. We propose transgressive hybrids may play an important role in population persistence under future warming conditions.

Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Histone derived antimicrobial peptides identified from Mytilus coruscus serum by peptidomics.

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus, and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine.

Multidrug resistant Vibrio spp. identified from mussels farmed for human consumption in Central Italy.

AIMS: This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. METHODS AND RESULTS: A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays. CONCLUSIONS: Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world".

Uranium contamination of bivalve Mytilus galloprovincialis, speciation and localization.

Uranium is a natural radioelement (also a model for heavier actinides), but may be released through anthropogenic activities. In order to assess its environmental impact in a given ecosystem, such as the marine system, it is essential to understand its distribution and speciation, and also to quantify its bioaccumulation. Our objective was to improve our understanding of the transfer and accumulation of uranium in marine biota with mussels taken here as sentinel species because of their sedentary nature and ability to filter seawater. We report here on the investigation of uranium accumulation, speciation, and localization in Mytilus galloprovincialis using a combination of several analytical (Inductively Coupled Plasma Mass Spectrometry, ICP-MS), spectroscopic (X ray Absorption Spectroscopy, XAS, Time Resolved Laser Induced Fluorescence Spectroscopy, TRLIFS), and imaging (Transmission Electron Microscopy, TEM, µ-XAS, Secondary Ion Mass Spectrometry, SIMS) techniques. Two cohorts of mussels from the Toulon Naval Base and the Villefranche-sur-Mer location were studied. The measurement of uranium Concentration Factor (CF) values show a clear trend in the organs of M. galloprovincialis: hepatopancreas â‰« gill > body ≥ mantle > foot. Although CF values for the entire mussel are comparable for TNB and VFM, hepatopancreas values show a significant increase in those from Toulon versus Villefranche-sur-Mer. Two organs of interest were selected for further spectroscopic investigations: the byssus and the hepatopancreas. In both cases, U(VI) (uranyl) is accumulated in a diffuse pattern, most probably linked to protein complexing functions, with the absence of a condensed phase. While such speciation studies on marine organisms can be challenging, they are an essential step for deciphering the impact of metallic radionuclides on the marine biota in the case of accidental release. Following our assumptions on uranyl speciation in both byssus and hepatopancreas, further steps will include the inventory and identification of the proteins or metabolites involved.

Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis).

BACKGROUND: Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. RESULTS: Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. CONCLUSIONS: We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

Marteilia pararefringens infections are more frequent than revealed by the Norwegian surveillance programme, highlighting the need for its improvement.

Norway had historically been considered free of marteiliosis in bivalves since the disease surveillance programme began in 1995. However, in 2016, Marteilia pararefringens, a protistan parasite of mussels Mytilus spp., was described in a heliothermic lagoon-a poll-previously used to produce flat oyster spat. To study whether the parasite was introduced, and possibly spread, via the historical flat oyster networks on the south and west coast, we sampled aquaculture polls that were part of different networks of farmers and wild, natural polls with no aquaculture activity. Additionally, we sampled mussel banks influenced by polls and sheltered bays that could have a similar environment to that of polls. We identified 7 sites with M. pararefringens-infected mussel populations: 5 were polls used in flat oyster production and 2 were in fjord areas with no known connection to any bivalve aquaculture. Prevalence ranged between 2 and 88%. At one site, Trysfjorden, we found M. pararefringens in atypical organs, including the gills, mantle, and intestine. Marteilia-like cells were also observed in the epithelium, lumen, and surrounding connective tissue of metanephridia and in the sinus of the anterior retractor muscle. Our results demonstrate that the parasite is more widespread than previously thought and is neither isolated to polls nor connected directly to aquaculture activity. Lastly, our findings highlight the need for an improved sampling strategy in surveillance programmes to detect marteiliosis in mussels.

Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Shell mineralogy of a foundational marine species, Mytilus californianus, over half a century in a changing ocean.

Anthropogenic warming and ocean acidification are predicted to negatively affect marine calcifiers. While negative effects of these stressors on physiology and shell calcification have been documented in many species, their effects on shell mineralogical composition remains poorly known, especially over longer time periods. Here, we quantify changes in the shell mineralogy of a foundation species, Mytilus californianus, under 60 y of ocean warming and acidification. Using historical data as a baseline and a resampling of present-day populations, we document a substantial increase in shell calcite and decrease in aragonite. These results indicate that ocean pH and saturation state, not temperature or salinity, play a strong role in mediating the shell mineralogy of this species and reveal long-term changes in this trait under ocean acidification.

How water acidification influences the organism antioxidant capacity and gill structure of Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) at normoxia and hypoxia.

The effect of water acidification in combination with normoxia or hypoxia on the antioxidant capacity and oxidative stress markers in gills and hemolymph of the Mediterranean mussel (Mytilus galloprovincialis), as well as on gill microstructure, has been evaluated through an in vivo experiment. Mussels were exposed to a low pH (7.3) under normal dissolved oxygen (DO) conditions (8 mg/L), and hypoxia (2 mg/L) for 8 days, and samples were collected on days 1, 3, 6, and 8 to evaluate dynamic changes of physiological responses. Cytoplasmic concentrations of reactive oxygen species (ROS) and levels of DNA damage were measured in hemocytes, while the activity of catalase (CAT) and superoxide dismutase (SOD) and histopathological changes were assessed in gills. The results revealed that while water acidification did not significantly affect the activity of SOD and CAT in gills under normoxic and hypoxic conditions, there was a trend towards suppression of CAT activity at the end of the experimental period (day 8). Similarly, we did not observe increased formation of ROS in hemocytes or changes in the levels of DNA damage during the experimental period. These results strongly suggest that the oxidative stress response system in mussels is relatively stable to experimental conditions of acidification and hypoxia. Experimental acidification under normoxia and hypoxia caused changes to the structure of the gills, leading to various histopathological alterations, including dilation, hemocyte infiltration into the hemal sinuses, intercellular edema, vacuolization of epithelial cells in gill filaments, lipofuscin accumulation, changes in the shape and adjacent gill filaments, hyperplasia, exfoliation of the epithelial layer, necrosis, swelling, and destruction of chitinous layers (chitinous rods). Most of these alterations were reversible, non-specific changes that represent a general inflammatory response and changes in the morphology of the gill filaments. The dynamics of histopathological alterations suggests an active adaptive response of gills to environmental stresses. Taken together, our data indicate that Mediterranean mussels have a relative tolerance to water acidification and hypoxia at tissue and cellular levels.

Survey on Perkinsus species in two economic mussels (Mytilus coruscus and M. galloprovincialis) along the coast of the East China Sea and the Yellow Sea.

Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest.

Chalcone derivatives as promising antifoulants: Molecular optimization, bioactivity evaluation and performance in coatings.

Marine biofouling remains a huge concern for maritime industries and for environmental health. Although the current biocide-based antifouling coatings can prevent marine biofouling, their use has been associated with toxicity for the marine environment, being urgent to find sustainable alternatives. Previously, our research group has identified a prenylated chalcone (1) with promising antifouling activity against the settlement of larvae of the macrofouling species Mytilus galloprovincialis (EC50 = 16.48 µM and LC50 > 200 µM) and lower ecotoxicity when compared to Econea®, a commercial antifouling agent in use. Herein, a series of chalcone 1 analogues were designed and synthesized in order to obtain optimized antifouling compounds with improved potency while maintaining low ecotoxicity. Compounds 8, 15, 24, and 27 showed promising antifouling activity against the settlement of M. galloprovincialis larvae, being dihydrochalcone 27 the most potent. The effect of compound 24 was associated with the inhibition of acetylcholinesterase activity. Among the synthesized compounds, compound 24 also showed potent complementary activity against Navicula sp. (EC50 = 4.86 µM), similarly to the lead chalcone 1 (EC50 = 6.75 µM). Regarding the structure-activity relationship, the overall results demonstrate that the substitution of the chalcone of the lead compound 1 by a dihydrochalcone scaffold resulted in an optimized potency against the settlement of mussel larvae. Marine polyurethane (PU)-based coatings containing the best performed compound concerning anti-settlement activity (dihydrochalcone 27) were prepared, and mussel larvae adherence was reduced compared to control PU coatings.

Contamination acts as a genotype-dependent barrier to gene flow, causing genetic erosion and fine-grained population subdivision in Mussels from the Strait of Istanbul.

This study provides evidence of fine-grained genetic structuring in Mediterranean mussels (Mytilus galloprovincialis) from the Strait of Istanbul, caused by barriers to gene flow via contaminant-mediated selection. In this study, mitochondrial D-loop sequences were analyzed in mussels from 8 localities, all less than 30 kilometers apart, with differing contaminant loads. The results were: 1) Intra-population genetic differentiation (ΦST) between sites with high and low contaminant loads was high (up to 0.459), even at distances of only a few kilometers. 2) Genetic diversity was negatively correlated with the contaminant load ("genetic erosion"). 3) There was evidence of selection, based on haplotype frequencies and neutrality tests (Tajima's D), with purifying selection at the most contaminated site and balancing selection at the least contaminated. 4) Genetic distance was not correlated with geographic distance (no isolation-by-distance), but was correlated with contaminant load at each site. 5) Population dendrograms and Bayesian estimators of migration indicated that gene flow between sites was affected by contamination. For the dendrograms of the sampling sites, the clades clustered according to contaminant load more than geographic distance. Overall, these results suggest that 1) contamination may serve as a genotype-dependent dispersal barrier (i.e., contamination may not affect total number of migrants, just the relative proportions of the haplotypes in the established immigrants), leading strong population differentiation over short distances, and 2) genetic erosion may occur by a combination of selection and altered patterns of haplotype-specific gene flow. These effects may be more pronounced in the Strait of Istanbul than in other locations because of the riverine nature and strong, uni-directional current of the strait.