Transcriptomic responses of Antarctic clam Laternula elliptica to nanoparticles, at single and combined exposures reveal ecologically relevant biomarkers.

In recent years micro- and nanoplastics and metal-oxide nanomaterials have been found in several environmental compartments. The Antarctic soft clam Laternula elliptica is an endemic Antarctic species having a wide distribution in the Southern Ocean. Being a filter-feeder, it could act as suitable bioindicator of pollution from nanoparticles also considering its sensitivity to various sources of stress. The present study aims to assess the impact of polystyrene nanoparticles (PS-NP) and the nanometal titanium-dioxide (n-TiO2) on genome-wide transcript expression of L. elliptica either alone and in combination and at two toxicological relevant concentrations (5 and 50 µg/L) during 96 h exposure. Transcript-target qRT-PCR was performed with the aim to identify suitable biomarkers of exposure and effects. As expected, at the highest concentration tested, the clustering was clearer between control and exposed clams. A total of 221 genes resulted differentially expressed in exposed clams and control ones, and 21 of them had functional annotation such as ribosomal proteins, antioxidant, ion transport (osmoregulation), acid-base balance, immunity, lipid metabolism, cell adhesion, cytoskeleton, apoptosis, chromatin condensation and cell signaling. At functional level, relevant transcripts were shared among some treatments and could be considered as general stress due to nanoparticle exposure. After applying transcript-target approach duplicating the number of clam samples, four ecologically relevant transcripts were revealed as biomarkers for PS-NP, n-TiO2 and their combination at 50 µg/L, that could be used for monitoring clams' health status in different Antarctic localities.

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.

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.

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.

Autophagic processes in Mytilus galloprovincialis hemocytes: Effects of Vibrio tapetis.

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.

Killing of Vibrio cholerae and Escherichia coli Strains Carrying D-mannose-sensitive Ligands by Mytilus Hemocytes is Promoted by a Multifunctional Hemolymph Serum Protein.

In aquatic environments, bivalve mollusks represent an important ecological niche for microorganisms. Persistence of bacteria in bivalve tissues partly depends on their capacity to survive the bactericidal activity of the hemolymph due to both cellular (hemocyes) and soluble serum factors (e.g., enzymes, lectins, opsonins). The extrapallial protein (EP) present in serum of Mytilus galloprovincialis (MgEP) has been recently shown to work as an opsonin promoting D-mannose sensitive (MS) interactions of the bivalve pathogen Vibrio aestuarianus 01/032 strain with the hemocytes. In this study, the role of MgEP in adhesion and killing of other bacteria carrying MS sensitive ligands was investigated. MgEP enhanced adhesion to and killing by hemocytes of Vibrio cholerae ElTor N16961, expressing the MS hemagglutin (MSHA), as well as of Escherichia coli MG1655, carrying type 1 fimbriae. These results further support the recent finding that the multifunctional MgEP also acts as an opsonin involved in mussel defense towards bacteria carrying MS ligands. In addition, these results contribute to elucidate the ecology of bacterial pathogens that can be transmitted to humans via shellfish consumption.

Combined effects of n-TiO2 and 2,3,7,8-TCDD in Mytilus galloprovincialis digestive gland: A transcriptomic and immunohistochemical study.

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. In the marine mussel Mytilus galloprovincialis, exposure to nanosized titanium dioxide (n-TiO2), one of the most widespread type of NPs in use, in combination with and 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD), chosen as model organic xenobiotic, was shown to induce significant changes in different biomarkers in hemocytes, gills and digestive gland, with distinct effects depending on cell/tissue and type of response measured. In this work, the interactive effects of n-TiO2 and TCDD at the tissue level were further investigated in mussel digestive gland using an integrated approach transcriptomics/immunohistochemistry. Mussels were exposed to n-TiO2 (100µgL(-1)) and TCDD (0.25µgL(-1)), alone and in combination, for 96h. Transcriptomic analysis identified 48-, 49- and 62 Differentially Expressed Genes (DEGs) in response to n-TiO2, TCDD and n-TiO2/TCDD, respectively. Gene Ontology (GO) term analysis revealed distinct biological processes affected in different experimental conditions. n-TiO2 mainly up-regulated cytoskeletal genes, while TCDD up-regulated endocrine and signal transduction related processes. Co-exposure induced transcriptional changes common to individual treatments, and identified a newly generated process, response to chemical stimulus. Transcription of selected genes was verified by qPCR. Moreover, expression of tubulin, as an example of target protein of interest identified by gene transcription data, was evaluated in tissue sections by immunolabelling. Tissue TCDD accumulation was evaluated by immunofluorescence with an anti-dioxins antibody. The results demonstrate both distinct and interactive effects of n-TiO2 and TCDD in mussel digestive gland at the molecular and tissue level, identify the main molecular targets involved, and underline how exposure to the n-TiO2/TCDD mixture does not result in increased TCDD accumulation and overall stressful conditions in the tissue. These represent the first data on transcriptional responses of marine invertebrates to exposure not only to n-TiO2 as a model of NP, but also to a legacy contaminant like TCDD.

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins.

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Neuroendocrine functions of monoamines in invertebrates: Focus on bivalve molluscs.

Monoamines (MA) such as serotonin, catecholamines (dopamine, norepinephrine, epinephrine), and trace amines (octopamine, tyramine), are neurotransmitters and neuroendocrine modulators in vertebrates, that contribute to adaptation to the environment. Although MA are conserved in evolution, information is still fragmentary in invertebrates, given the diversity of phyla and species. However, MA are crucial in homeostatic processes in these organisms, where the absence of canonical endocrine glands in many groups implies that the modulation of physiological functions is essentially neuroendocrine. In this review, we summarize available information on MA systems in invertebrates, with focus on bivalve molluscs, that are widespread in different aquatic environments, where they are subjected to a variety of environmental stimuli. Available data are reviewed on the presence of the different MA in bivalve tissues, their metabolism, target cells, signaling pathways, and the physiological functions modulated in larval and adult stages. Research gaps and perspectives are highlighted, in order to enrich the framework of knowledge on MA neuroendocrine functions, and on their role in adaptation to ongoing and future environmental changes.

Exposure to nanoplastics and nanomaterials either single and combined affects the gill-associated microbiome of the Antarctic soft-shelled clam Laternula elliptica.

Nanoplastics and engineering nanomaterials (ENMs) are contaminants of emerging concern (CECs), increasingly being detected in the marine environment and recognized as a potential threat for marine biota at the global level including in polar areas. Few studies have assessed the impact of these anthropogenic nanoparticles in the microbiome of marine invertebrates, however combined exposure resembling natural scenarios has been overlooked. The present study aimed to evaluate the single and combined effects of polystyrene nanoparticles (PS NP) as proxy for nanoplastics and nanoscale titanium dioxide (nano-TiO2) on the prokaryotic communities associated with the gill tissue of the Antarctic soft-shell clam Laternula elliptica, a keystone species of marine benthos Wild-caught specimens were exposed to two environmentally relevant concentrations of carboxylated PS NP (PS-COOH NP, ∼62 nm size) and nano-TiO2 (Aeroxide P25, ∼25 nm) as 5 and 50 µg/L either single and combined for 96h in a semi-static condition.Our findings show a shift in microbiome composition in gills of soft-shell clams exposed to PS NP and nano-TiO2 either alone and in combination with a decrease in the relative abundance of OTU1 (Spirochaetaceae). In addition, an increase of gammaproteobacterial OTUs affiliated to MBAE14 and Methylophagaceae (involved in ammonia denitrification and associated with low-quality water), and the OTU Colwellia rossensis (previously recorded in polluted waters) was observed. Our results suggest that nanoplastics and nano-TiO2 alone and in combination induce alterations in microbiome composition by promoting the increase of negative taxa over beneficial ones in the gills of the Antarctic soft-shell clam. An increase of two low abundance OTUs in PS-COOH NPs exposed clams was also observed. A predicted gene function analysis revealed that sugar, lipid, protein and DNA metabolism were the main functions affected by either PS-COOH NP and nano-TiO2 exposure. The molecular functions involved in the altered affiliated OTUs are novel for nano-CEC exposures.

The biocide triclosan as a potential developmental disruptor in Mytilus early larvae.

The broadly utilized biocide triclosan (TCS) is continuously discharged in water compartments worldwide, where it is detected at concentrations of ng-µg/L. Given its lipophilicity and bioaccumulation, TCS is considered potentially harmful to human and environmental health and also as a potential endocrine disruptor (ED) in different species. In aquatic organisms, TCS can induce a variety of effects: however, little information is available on its possible impact on invertebrate development. Early larval stages of the marine bivalve Mytilus galloprovincialis have been shown to be sensitive to environmental concentrations of a number of emerging contaminants, including EDs. In this work, the effects of TCS were first evaluated in the 48 h larval assay in a wide concentration range (0.001-1,000 µg/L). TCS significantly affected normal development of D-veligers (LOEC = 0.1 µg/L; EC50 = 236.1 µg/L). At selected concentrations, the mechanism of action of TCS was investigated. TCS modulated transcription of different genes involved in shell mineralization, endocrine signaling, ceramide metabolism, and biotransformation, depending on larval stage (24 and 48 h post-fertilization-hpf) and concentration (1 and 10 µg/L). At 48 hpf and 10 µg/L TCS, calcein staining revealed alterations in CaCO3 deposition, and polarized light microscopy showed the absence of shell birefringence due to the mineralized phase. Observations by scanning electron microscopy highlighted a variety of defects in shell formation from concentrations as low as 0.1 µg/L. The results indicate that TCS, at environmental exposure levels, can act as a developmental disruptor in early mussel larvae mainly by interfering with the processes of biomineralization.

Changes in phospholipid profiles in early larval stages of the marine mussel Mytilus galloprovincialis indicate a role of ceramides in bivalve development.

BACKGROUND: Phospholipids are highly diverse molecules with pleiotropic biological roles, from membrane components and signaling molecules, whose composition can change in response to both endogenous and external stimuli. Recent lipidomic studies on edible bivalve mollusks were focused on lipid nutritional value and growth requirements. However, no data are available on phospholipid profiles during bivalve larval development. In the model marine bivalve Mytilus galloprovincialis, early larvae (up to 48 hours post fertilization-hpf) undergo dramatic molecular and functional changes, including shell biogenesis and neurogenesis, that are sustained by egg lipid reserves. Changes in phospholipid composition may also occur participating in the complex processes of early development. OBJECTIVE: The lipidome of M. galloprovincialis eggs and early larval stages (24 and 48 hpf) was investigated in order to identify possible changes in phospholipid classes and related metabolic pathways that may play a role in key steps of development. MATERIALS AND METHODS: Lipidomic analysis were performed by NMR spectroscopy and liquid chromatography-mass spectrometry (LC-MS), with focus on phospholipids. Shifts in relative species composition of phosphatidylcholine, phosphatidylethanolamine, plasmalogen, and ceramide aminoethylphosphonate-CAEP, the bivalve analogue of the main mammalian ceramide sphingomyelin, were observed. Expression of genes involved in ceramide homeostasis was also modulated from eggs to early larval stages. RESULTS: The results represent the first data on changes in phospholipid composition in bivalve larvae and suggest a functional role of phospholipids in mussel early development. CONCLUSION: The results underline the importance of lipidomic studies in bivalve larvae, in both physiological conditions and in response to environmental stress.

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.

Physiological Roles of Serotonin in Bivalves: Possible Interference by Environmental Chemicals Resulting in Neuroendocrine Disruption.

Contaminants of Emerging Concerns (CECs) are defined as chemicals not commonly monitored in aquatic ecosystems, but with the potential to cause adverse effects on biota. CECs include Endocrine Disrupting Chemicals (EDCs) and Neuro-Endocrine disruptors (NEDs) of vertebrates. However, most invertebrates only rely on neuroendocrine systems to maintain homeostatic processes. Although conserved neuroendocrine components have been characterized in ecologically relevant groups, limited knowledge on invertebrate neuroendocrinology makes it difficult to define EDCs and NEDs in most species. The monoamine serotonin (5-hydroxytryptamine, 5-HT) acts both as a neurotransmitter and as a peripheral hormone in mammals. In molluscs, 5-HT is involved in multiple physiological roles and molecular components of the serotonergic system have been identified. This review is focused on the effects of CECs on the serotonergic system of bivalve molluscs. Bivalves are widespread in all aquatic environments, estuarine and coastal areas in particular, where they are exposed to a variety of chemicals. In bivalves, 5-HT is involved in gametogenesis and spawning, oocyte maturation and sperm motility, regulates heart function, gill ciliary beating, mantle/siphon function, the ''catch'' state of smooth muscle and immune responses. Components of 5-HT transduction (receptors and signaling pathways) are being identified in several bivalve species. Different CECs have been shown to affect bivalve serotonergic system. This particularly applies to antidepressants, among the most commonly detected human pharmaceuticals in the aquatic environment. In particular, selective serotonin reuptake inhibitors (SSRIs) are frequently detected in seawater and in bivalve tissues. Information available on the effects and mechanisms of action of SSRIs on the serotonergic system of adult bivalves is summarized. Data are also reported on the effects of CECs on development of neuroendocrine pathways of early larval stages, in particular on the effects of model EDCs in the marine mussel Mytilus galloprovincialis. Overall, available data point at the serotonergic system as a sensitive target for neuroendocrine disruption in bivalves. The results contribute drawing Adverse Outcome Pathways (AOPs) for model EDCs and SSRIs in larvae and adults. However, basic research on neuroendocrine signaling is still needed to evaluate the potential impact of neuroendocrine disruptors in key invertebrate groups of aquatic ecosystems.

Soluble mediators of innate immunity in annelids and bivalve mollusks: A mini-review.

Annelids and mollusks, both in the superphylum of Lophotrochozoa (Bilateria), are important ecological groups, widespread in soil, freshwater, estuarine, and marine ecosystems. Like all invertebrates, they lack adaptive immunity; however, they are endowed with an effective and complex innate immune system (humoral and cellular defenses) similar to vertebrates. The lack of acquired immunity and the capacity to form antibodies does not mean a lack of specificity: invertebrates have evolved genetic mechanisms capable of producing thousands of different proteins from a small number of genes, providing high variability and diversity of immune effector molecules just like their vertebrate counterparts. This diversity allows annelids and mollusks to recognize and eliminate a wide range of pathogens and respond to environmental stressors. Effector molecules can kill invading microbes, reduce their pathogenicity, or regulate the immune response at cellular and systemic levels. Annelids and mollusks are "typical" lophotrochozoan protostome since both groups include aquatic species with trochophore larvae, which unite both taxa in a common ancestry. Moreover, despite their extensive utilization in immunological research, no model systems are available as there are with other invertebrate groups, such as Caenorhabditis elegans or Drosophila melanogaster, and thus, their immune potential is largely unexplored. In this work, we focus on two classes of key soluble mediators of immunity, i.e., antimicrobial peptides (AMPs) and cytokines, in annelids and bivalves, which are the most studied mollusks. The mediators have been of interest from their first identification to recent advances in molecular studies that clarified their role in the immune response.

Ceramide Aminoethylphosphonate as a New Molecular Target for Pore-Forming Aegerolysin-Based Protein Complexes.

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE. This pore formation is the main underlying molecular mechanism of potent and selective insecticidal activity of OlyA6/PlyB complexes against two economically important coleopteran plant pests: the western corn rootworm and the Colorado potato beetle. In contrast to insects, the main sphingolipid in cell membranes of marine invertebrates (i.e., molluscs and cnidarians) is ceramide aminoethylphosphonate (CAEP), a CPE analogue built on a phosphono rather than the usual phosphate group in its polar head. Our targeted lipidomic analyses of the immune cells (hemocytes) of the marine bivalve, the mussel Mytilus galloprovincialis, confirmed the presence of 29.0 mol% CAEP followed by 36.4 mol% of phosphatidylcholine and 34.6 mol% of phosphatidylethanolamine. Further experiments showed the potent binding of OlyA6 to artificial lipid vesicles supplemented with mussel CAEP, and strong lysis of these vesicles by the OlyA6/PlyB mixture. In Mytilus haemocytes, short term exposure (max. 1 h) to the OlyA6/PlyB mixture induced lysosomal membrane destabilization, decreased phagocytic activity, increased Annexin V binding and oxyradical production, and decreased levels of reduced glutathione, indicating rapid damage of endo-lysosomal and plasma membranes and oxidative stress. Our data suggest CAEP as a novel high-affinity receptor for OlyA6 and a target for cytolytic OlyA6/PlyB complexes.

Immunological Responses of Marine Bivalves to Contaminant Exposure: Contribution of the -Omics Approach.

The increasing number of data studies on the biological impact of anthropogenic chemicals in the marine environment, together with the great development of invertebrate immunology, has identified marine bivalves as a key invertebrate group for studies on immunological responses to pollutant exposure. Available data on the effects of contaminants on bivalve immunity, evaluated with different functional and molecular endpoints, underline that individual functional parameters (cellular or humoral) and the expression of selected immune-related genes can distinctly react to different chemicals depending on the conditions of exposure. Therefore, the measurement of a suite of immune biomarkers in hemocytes and hemolymph is needed for the correct evaluation of the overall impact of contaminant exposure on the organism's immunocompetence. Recent advances in -omics technologies are revealing the complexity of the molecular players in the immune response of different bivalve species. Although different -omics represent extremely powerful tools in understanding the impact of pollutants on a key physiological function such as immune defense, the -omics approach has only been utilized in this area of investigation in the last few years. In this work, available information obtained from the application of -omics to evaluate the effects of pollutants on bivalve immunity is summarized. The data shows that the overall knowledge on this subject is still quite limited and that to understand the environmental relevance of any change in immune homeostasis induced by exposure to contaminants, a combination of both functional assays and cutting-edge technology (transcriptomics, proteomics, and metabolomics) is required. In addition, the utilization of metagenomics may explain how the complex interplay between the immune system of bivalves and its associated bacterial communities can be modulated by pollutants, and how this may in turn affect homeostatic processes of the host, host-pathogen interactions, and the increased susceptibility to disease. Integrating different approaches will contribute to knowledge on the mechanism responsible for immune dysfunction induced by pollutants in ecologically and economically relevant bivalve species and further explain their sensitivity to multiple stressors, thus resulting in health or disease.

Comparison of Different Commercial Nanopolystyrenes: Behavior in Exposure Media, Effects on Immune Function and Early Larval Development in the Model Bivalve Mytilus galloprovincialis.

In the absence of standard methods for the detection/quantification of nanoplastics (NPs) in environmental samples, commercial nanopolymers are utilized as proxies for toxicity testing and environmental risk assessment. In marine species, a considerable amount of data are now available on the effects of nanopolystyrene (PS-NPs) of different size/surface characteristics. In this work, amino modified PS-NPs (PS-NH2) (50 and 100 nm), purchased from two different companies, were compared in terms of behavior in exposure media and of biological responses, from molecular to organism level, in the model marine bivalve Mytilus. Different PS-NH2 showed distinct agglomeration and surface charge in artificial sea water (ASW) and hemolymph serum (HS). Differences in behavior were largely reflected by the effects on immune function in vitro and in vivo and on early larval development. Stronger effects were generally observed with PS-NH2 of smaller size, showing less agglomeration and higher positive charge in exposure media. Specific molecular interactions with HS components were investigated by the isolation and characterization of the NP-corona proteins. Data obtained in larvae demonstrate interference with the molecular mechanisms of shell biogenesis. Overall, different PS-NH2 can affect the key physiological functions of mussels at environmental concentrations (10 µg/L). However, detailed information on the commercial NPs utilized is required to compare their biological effects among laboratory experiments.

Conservation of Cell Communication Systems in Invertebrate Host-Defence Mechanisms: Possible Role in Immunity and Disease.

Innate immunity is continuously revealing multiple and highly conserved host-defence mechanisms. Studies on mammalian immunocytes are showing different communication systems that may play a role in coordinating innate immune responses also in invertebrates. Extracellular traps (ETs) are an immune response by which cells release net-like material, including DNA, histones and proteins. ETs are thought to immobilise and kill microorganisms, but are also involved in inflammation and autoimmune disease. Immune cells are also known to communicate through extracellular vesicles secreted in the extracellular environment or exosomes, which can carry a variety of different signalling molecules. Tunnelling nanotubes (TNTs) represent a direct cell-to-cell communication over a long distance, that allow for bi- or uni-directional transfer of cellular components between cells. Their functional role in a number of physio-pathological processes, including immune responses and pathogen transfer, has been underlined. Although ETs, exosomes, and TNTs have been described in invertebrate species, their possible role in immune responses is not fully understood. In this work, available data on these communication systems are summarised, in an attempt to provide basic information for further studies on their relevance in invertebrate immunity and disease.

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.