Cirsiliol and Quercetin Inhibit ATP Synthesis and Decrease the Energy Balance in Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Resistant Staphylococcus epidermidis (MRSE) Strains Isolated from Patients.

Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit F1Fo-ATP synthase (ATP synthase) and that bacteria need a constant energy production to maintain their homeostasis, we evaluated the effect of two flavones, cirsiliol (tri-hy-droxy-6,7-dimethoxyflavone) and quercetin (3,3,4,5,7-pentahydroxyflavone), on energy production and intracellular ATP content in a methicillin-resistant Staphylococcus aureus (MRSA) strain and a methicillin-resistant Staphylococcus epidermidis (MRSE) strain isolated from patients, comparing the results to those obtained by treating the bacteria with oligomycin, a specific ATP synthase Fo moiety inhibitor. Real-time quantitative ATP synthesis and total ATP content of permeabilized Gram-positive bacteria were assayed by luminometry. The results showed that cirsiliol and quercetin inhibited ATP synthase and decreased the intracellular ATP levels in both strains, although the effect was higher in MRSE. In addition, while cirsiliol and quercetin acted immediately after the treatment, oligomycin inhibited ATP synthesis only after 30 min of incubation, suggesting that the different responses may depend on the different permeability of the bacterial wall to the three molecules. Thus, cirsiliol and quercetin could be considered potential additions to antibiotics due to their ability to target ATP synthase, against which bacteria cannot develop resistance.

Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

2D Collagen Membranes from Marine Demosponge Chondrosia reniformis (Nardo, 1847) for Skin-Regenerative Medicine Applications: An In Vitro Evaluation.

Research in tissue engineering and regenerative medicine has an ever-increasing need for innovative biomaterials suitable for the production of wound-dressing devices and artificial skin-like substitutes. Marine collagen is one of the most promising biomaterials for the production of such devices. In this study, for the first time, 2D collagen membranes (2D-CMs) created from the extracellular matrix extract of the marine demosponge Chondrosia reniformis have been evaluated in vitro as possible tools for wound healing. Fibrillar collagen was extracted from a pool of fresh animals and used for the creation of 2D-CMs, in which permeability to water, proteins, and bacteria, and cellular response in the L929 fibroblast cell line were evaluated. The biodegradability of the 2D-CMs was also assessed by following their degradation in PBS and collagenase solutions for up to 21 days. Results showed that C. reniformis-derived membranes avoided liquid and protein loss in the regeneration region and also functioned as a strong barrier against bacteria infiltration into a wound. Gene expression analyses on fibroblasts stated that their interaction with 2D-CMs is able to improve fibronectin production without interfering with the regular extracellular matrix remodeling processes. These findings, combined with the high extraction yield of fibrillar collagen obtained from C. reniformis with a solvent-free approach, underline how important further studies on the aquaculture of this sponge could be for the sustainable production and biotechnological exploitation of this potentially promising and peculiar biopolymer of marine origin.

Large-scale impact of the 2016 Marine Heatwave on the plankton-associated microbial communities of the Great Barrier Reef (Australia).

The Great Barrier Reef (GBR) is the world's largest coral ecosystem and is threatened by climate change. This study investigated the impact of the 2016 Marine Heatwave (MHW) on plankton associated microbial communities along a ∼800 km transect in the GBR. 16S rRNA gene metabarcoding of archived plankton samples collected from November 2014 to August 2016 in this region showed a significant increase in Planctomycetes and bacteria belonging to the genus Vibrio and Synechococcus during and after the heatwave. Notably, Droplet Digital PCR and targeted metagenomic analysis applied on samples collected four months after the MHW event revealed the presence of several potential pathogenic Vibrio species previously associated with diseases in aquatic animals. Overall, the 2016 MHW significantly impacted the surface picoplankton community and fostered the spread of potentially pathogenic bacteria across the GBR providing an additional threat for marine biodiversity in this area.

Searching pathogenic bacteria in the rare biosphere of the ocean.

Harmful marine bacteria, such as Vibrio or Aeromonas species, typically exist at low abundance in ocean environments but represent a reservoir from which epidemics can arise. Particularly, Vibrio strains and their associated infections are on the rise globally due to increasing sea surface temperature representing an emergent threat for human and animal health also being responsible for large economic losses in the aquaculture industry worldwide. New technological approaches are needed to improve strategies targeting these pathogens. This review discusses new approaches based on improved sampling strategies and novel analytical methods offering increased accuracy, high throughput, and informativeness to study and detect microbial pathogens in the marine environment. Detecting and characterizing ultra-low-abundance pathogenic strains can serve as a critical tool in risk management and outbreak prevention of diseases caused by emerging marine pathogens.

Responses of Mytilus galloprovincialis to challenge with environmental isolates of the potential emerging pathogen Malaciobacter marinus.

Bacteria of the Arcobacter-like spp. represent emerging foodborne zoonotic pathogens in humans and animals. Their increasing presence in seafood, suggesting higher occurrence in seawater due to marine pollution, is raising some environmental concern. Although Arcobacter is frequently detected in diseased oysters and stressed bivalve species, no data are available so far on its potential pathogenicity or interactions with the immune system of the bivalve host. In this work, responses to challenge with two strains of Malaciobacter marinus IRTA-19-131 and IRTA-19-132, R1 and R2), isolated from adult Crassostrea gigas during a mortality event in 2019 in Spain, were investigated in the mussel Mytilus galloprovincialis. In vivo experiments were performed in larvae (48 h post-fertilization), and in adult mussels at 24 h post-injection, in order to evaluate the pathogenicity for early developmental stages, and the hemolymph immune responses, respectively. Both R1 and R2 were moderately pathogenic to early larvae, with significant decreases in the development of normal D-veligers from 104 and 103 CFU/mL, respectively. In adults, both strains decreased hemocyte lysosomal membrane stability (LMS), and stimulated extracellular defense responses (ROS production and lysozyme activity). The interactions between mussel hemocytes and M. marinus were investigated in in vitro short-term experiments (30-90 min) using the R1 strain (106-108 CFU/mL). R1 decreased LMS and induced lysosomal enlargement, but not cell detachment or death, and stimulated extracellular ROS production and lysozyme release, confirming in vivo data. Moreover, lysosomal internalization and degradation of bacteria were observed, together with changes in levels of activated mTor and LC3, indicating phagocytic activity. Overall, the results indicate the activation of both extracellular and intracellular immune defenses against M. marinus R1. Accordingly, these responses resulted in a significant hemolymph bactericidal activity, with a large contribution of hemolymph serum. The results represent the first data on the potential pathogenicity of Arcobacter isolated from a shellfish mortality to bivalve larvae and adults, and on their interactions with the immune system of the host.

Recent advances in bivalve-microbiota interactions for disease prevention in aquaculture.

In bivalves, no clear-cut functional role of microbiota has yet been identified, although many publications suggest that they could be involved in nutrition or immunity of their host. In the context of climate change, integrative approaches at the crossroads of disciplines have been developed to explore the environment-host-pathogen-microbiota system. Here, we attempt to synthesize work on (1) the current methodologies to analyse bivalve microbiota, (2) the comparison of microbiota between species, between host compartments and their surrounding habitat, (3) how the bivalve microbiota are governed by environmental factors and host genetics and (4) how host-associated microorganisms act as a buffer against pathogens and/or promote recovery, and could thereby play a role in the prevention of disease or mortalities.

Continuous Plankton Recorder in the omics era: from marine microbiome to global ocean observations.

First routinely deployed in 1931 the Continuous Plankton Recorder (CPR) technology has established the most extensive, marine biological sampling programme in the world. With more than 90 years of sampling, over a total of 8 million nautical miles covered and 500 000 curated samples, the CPR survey provides a gold mine of information available to marine researchers. Such information is likely to exponentially increase thanks to new cutting-edge molecular technologies that are beginning to be applied on CPR samples. In this review we aim to address the exciting developments that the genomic revolution is having on ​CPR applications from the study of marine microbiome to ocean plankton communities leading to ​a new 'digital era' of the global ocean CPR observation programme.

A deep-sea bacterium related to coastal marine pathogens.

Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here, we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309 m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptation to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infections in farmed oysters. The results of functional in vitro assays on immunocytes (haemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, which might have played an evolutionary role in the origin of coastal marine pathogens.

Frenemies: Interactions between Rhizospheric Bacteria and Fungi from Metalliferous Soils.

Is it possible to improve the efficiency of bioremediation technologies? The use of mixed cultures of bacteria and fungi inoculated at the rhizosphere level could promote the growth of the associated hyperaccumulating plant species and increase the absorption of metals in polluted soils, broadening new horizons on bioremediation purposes. This work investigates interactions between Ni-tolerant plant growth-promoting bacteria and fungi (BF) isolated from the rhizosphere of a hyperaccumulating plant. The aim is to select microbial consortia with synergistic activity to be used in integrated bioremediation protocols. Pseudomonas fluorescens (Pf), Streptomyces vinaceus (Sv) Penicilliumochrochloron (Po), and Trichoderma harzianum group (Th) were tested in mixes (Po-Sv, Po-Pf, Th-Pf, and Th-Sv). These strains were submitted to tests (agar overlay, agar plug, and distance growth co-growth tests), tailored for this aim, on Czapek yeast agar (CYA) and tryptic soy agar (TSA) media and incubated at 26 ± 1 °C for 10 days. BF growth, shape of colonies, area covered on plate, and inhibition capacity were evaluated. Most BF strains still exhibit their typical characters and the colonies separately persisted without inhibition (as Po-Sv) or with reciprocal confinement (as Th-Sv and Th-Pf). Even if apparently inhibited, the Po-Pf mix really merged, thus obtaining morphological traits representing a synergic co-growth, where both strains reached together the maturation phase and developed a sort of mixed biofilm. Indeed, bacterial colonies surround the mature fungal structures adhering to them without any growth inhibition. First data from in vivo experimentation with Po and Pf inocula in pot with metalliferous soils and hyperaccumulator plants showed their beneficial effect on plant growth. However, there is a lack of information regarding the effective co-growth between bacteria and fungi. Indeed, several studies, which directly apply the co-inoculum, do not consider suitable microorganisms consortia. Synergic rhizosphere BFs open new scenarios for plant growth promotion and soil bioremediation.

Linezolid Resistance Genes in Enterococci Isolated from Sediment and Zooplankton in Two Italian Coastal Areas.

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs.IMPORTANCE Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria; therefore, the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA, and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples from two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environments could reflect their spillover from human and/or animal reservoirs and could indicate that coastal seawaters also might represent a source of these resistance genes.

Antarctic coastal nanoplankton dynamics revealed by metabarcoding of desalination plant filters: Detection of short-term events and implications for routine monitoring.

One of the main requirements of any sound biological monitoring is the availability of long term and, possibly, temporal data with a high resolution. This is often difficult to be achieved, especially in Antarctica, due to a variety of logistic constraints, which make continuous sampling and monitoring activities generally unfeasible. Here we focus on the 5 µm filters used in the desalination plant of the Italian research base "Mario Zucchelli" in the Terra Nova Bay area (Ross Sea, Antarctica) to evaluate intra-annual coastal nanoplankton dynamics. These filters, together with others of larger mesh sizes, are used to decrease the amount of organisms and debris in the input seawater before the desalination processes take place, hence automatically collect the plankton present in the water column around the desalination system intake. We have used a DNA metabarcoding approach to characterize the communities retained by filters' sets collected in January 2012 and 2013. Intra-annual dynamics were disclosed with an unprecedented detail, that would not have been possible by using standard sampling approaches, and highlighted the importance of extreme, stochastic events such as katabatic wind pulses, which triggered dramatic, short-term shifts in coastal nanoplankton composition. This method, by combining a cost-effective sampling and molecular techniques, may represent a viable solution for long-term monitoring programs focusing on Antarctic coastal communities.

Aquatic reservoir of Vibrio cholerae in an African Great Lake assessed by large scale plankton sampling and ultrasensitive molecular methods.

The significance of large tropical lakes as environmental reservoirs of Vibrio cholerae in cholera endemic countries has yet to be established. By combining large scale plankton sampling, microbial culture and ultrasensitive molecular methods, namely Droplet Digital PCR (ddPCR) and targeted genomics, the presence of Vibrio cholerae was investigated in a 96,600 L volume of surface water collected on a 322 nautical mile (596 km) transect in Lake Tanganyika. V. cholerae was detected and identified in a large area of the lake. In contrast, toxigenic strains of V. cholerae O1 or O139 were not detected in plankton samples possibly in relation to environmental conditions of the lake ecosystem, namely very low salinity compared to marine brackish and coastal environments. This represents to our knowledge, the largest environmental study to determine the role of tropical lakes as a reservoir of V. cholerae.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

Impact of nanoplastics on hemolymph immune parameters and microbiota composition in Mytilus galloprovincialis.

Ocean contamination by micro- and nanoplastics represents a potential threat to marine biota, from bacterial communities to higher organisms. In this work, the effect of in vivo exposure of Mytilus galloprovincialis to amino modified nanopolystyrene (PS-NH2) (10 µg/L, 96 h) on hemolymph immune parameters and microbiota composition were investigated. Nanoplastics significantly affected immune parameters (decreased phagocytosis, increased ROS and lysozyme activity, inhibition of NO production). These changes were associated with a shift in hemolymph microbiota composition, with increase in some genera (Arcobacter-like, Psychrobium, Vibrio), and decreases in others (Shewanella, Mycoplasma). The results indicate that exposure to nanoplastics can impact on the microbiome of marine bivalves, and suggest that downregulation of immune defences induced by PS-NH2 may favour potentially pathogenic bacteria. These data underline how exposure to nanoplastics may represent a potential threat to the complex interplay between innate immunity and host microbiota, thus affecting the homeostatic processes involved in maintenance of organism health.

Shift in Immune Parameters After Repeated Exposure to Nanoplastics in the Marine Bivalve Mytilus.

Bivalves are widespread in coastal environments subjected to a wide range of environmental fluctuations: however, the rapidly occurring changes due to several anthropogenic factors can represent a significant threat to bivalve immunity. The mussel Mytilus spp. has extremely powerful immune defenses toward different potential pathogens and contaminant stressors. In particular, the mussel immune system represents a significant target for different types of nanoparticles (NPs), including amino-modified nanopolystyrene (PS-NH2) as a model of nanoplastics. In this work, the effects of repeated exposure to PS-NH2 on immune responses of Mytilus galloprovincialis were investigated after a first exposure (10 µg/L; 24 h), followed by a resting period (72-h depuration) and a second exposure (10 µg/L; 24 h). Functional parameters were measured in hemocytes, serum, and whole hemolymph samples. In hemocytes, transcription of selected genes involved in proliferation/apoptosis and immune response was evaluated by qPCR. First exposure to PS-NH2 significantly affected hemocyte mitochondrial and lysosomal parameters, serum lysozyme activity, and transcription of proliferation/apoptosis markers; significant upregulation of extrapallial protein precursor (EPp) and downregulation of lysozyme and mytilin B were observed. The results of functional hemocyte parameters indicate the occurrence of stress conditions that did not however result in changes in the overall bactericidal activity. After the second exposure, a shift in hemocyte subpopulations, together with reestablishment of basal functional parameters and of proliferation/apoptotic markers, was observed. Moreover, hemolymph bactericidal activity, as well as transcription of five out of six immune-related genes, all codifying for secreted proteins, was significantly increased. The results indicate an overall shift in immune parameters that may act as compensatory mechanisms to maintain immune homeostasis after a second encounter with PS-NH2.

Vibrio-bivalve interactions in health and disease.

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio-bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.