An apicomplexan parasite drives the collapse of the bay scallop population in New York.

The bay scallop, Argopecten irradians, represents a commercially, culturally and ecologically important species found along the United States' Atlantic and Gulf coasts. Since 2019, scallop populations in New York have been suffering large-scale summer mortalities resulting in 90-99% reduction in biomass of adult scallops. Preliminary investigations of these mortality events showed 100% prevalence of an apicomplexan parasite infecting kidney tissues. This study was designed to provide histological, ultrastructural and molecular characteristics of a non-described parasite, member of the newly established Marosporida clade (Apicomplexa) and provisionally named BSM (Bay Scallop Marosporida). Molecular diagnostics tools (quantitative PCR, in situ hybridization) were developed and used to monitor disease development. Results showed that BSM disrupts multiple scallop tissues including kidney, adductor muscle, gill, and gonad. Microscopy observations allowed the identification of both intracellular and extracellular stages of the parasite. Field surveys demonstrated a strong seasonal signature in disease prevalence and intensity, as severe cases and mortality increase as summer progresses. These results strongly suggest that BSM infection plays a major role in the collapse of bay scallop populations in New York. In this framework, BSM may synergistically interact with stressful environmental conditions to impair the host and lead to mortality.

Silver nanoparticles' impact on the gene expression of the cytosolic adaptor MyD-88 and the interferon regulatory factor IRF in the gills and digestive gland of mytilus galloprovincialis.

Silver nanoparticles (AgNPs) have been reported as stressors for the bivalves' immune system at different regulatory levels, impacting the detection step and receptors, and other mediators, as well as effector molecules. However, studies on how AgNPs impact the transmission of signals from receptors and whether they have an effect on mediators and transcription factors are still scarce. This study aims to investigate the effect of 12 hours of in vivo exposure to 100 µg/L of AgNPs on the gene expression of the cytosolic adaptor Myeloid, the differentiation protein 88 (MgMyD88-b), and the interferon regulatory factor (Me4-IRF) in the gills and digestive gland of Mytilus galloprovincialis, before and after blocking two major uptake pathways of nanoparticles (clathrin- and caveolae-mediated endocytosis). The results illustrate a tissue-specific gene expression of the MgMyD88-b and the Me4-IRF in the gills and digestive gland of M. galloprovincialis. In the gills, AgNPs did not significantly impact the expression of the two genes. However, blocking the caveolae-mediated endocytosis decreased the expression of Me4-IRF. However, inhibition of clathrin-mediated endocytosis in the digestive gland recorded a significant decrease in the expression of MgMyD88-b. Overall, the inhibition of the AgNPs' uptake routes have highlighted their potential interference with the immune response through the studied mediators' genes, which need to be studied further in future investigations.

A Comprehensive Review on Crustaceans' Immune System With a Focus on Freshwater Crayfish in Relation to Crayfish Plague Disease.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci, the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans' immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

Immunity in mussels: An overview of molecular components and mechanisms with a focus on the functional defenses.

Bivalves' immunity has received much more attention in the last decade, which resulted to a valuable growth in the availability of its molecular components. Such data availability coupled with the economical importance of these organisms aimed to shift the increase in the number of immunological and stress-related studies. Unfortunately, the crowd of generated data deciphering the involved physiological processes, investigators' differential conceptualization and the aimed objectives, has complicated the sensu stricto outlining of immune-related mechanisms. Overall, this review tried to compiles a summary about the molecular components of the mussels' immune response, surveying an overview of the mussels' functional immunity through gathering the most recent-related topics of bivalves' immunity as apoptosis and autophagy which deserves a great attention as stress-related mechanisms, the disseminated neoplasia as outbreak transmissible disease, not only within the same specie but also among different species, the hematopoiesis as topic that still generating interesting debate in the scientific community, the mucosal immunity described as the interface where host-pathogen interactions would occurs and determinate the late immune response, and innate immune memory and transgenerational priming, which described as very recent research topic with extensive applications in shellfish farming industry.

Redox proteomic insights into involvement of clathrin-mediated endocytosis in silver nanoparticles toxicity to Mytilus galloprovincialis.

Clathrin-mediated endocytosis is a major mode of nanoparticle (NP) internalization into cells. However, influence of internalization routes on nanoparticle toxicity is poorly understood. Here, we assess the impact of blocking clathrin-mediated endocytosis upon silver NP (AgNP) toxicity to gills and digestive glands of the mussel Mytilusgalloprovincialisusing the uptake inhibitor, amantadine. Animals were exposed for 12h to AgNP (< 50 nm) in the presence and absence of amantadine. Labeling of oxidative protein modifications, either thiol oxidation, carbonyl formation or both in two-dimensional electrophoresis separations revealed 16 differentially affected abundance spots. Amongst these, twelve hypothetical proteins were successfully identified by peptide mass fingerprinting (MALDI TOF-MS/MS). The proteins identified are involved in buffering redox status or in cytoprotection. We conclude that blockade of clathrin-mediated endocytosis protected against NP toxicity, suggesting this uptake pathway facilitates toxicity. Lysosomal degradation and autophagy are major mechanisms that might be induced to mitigate NP toxicity.

Role of endocytotic uptake routes in impacting the ROS-related toxicity of silver nanoparticles to Mytilus galloprovincialis: A redox proteomic investigation.

Oxidative stress is often implicated in nanoparticle toxicity. Several studies have highlighted the role of internalization routes in determining nanotoxicity. Here, we investigate how two endocytotic mechanisms (clathrin- and caveolae-mediated) impact on redox balance in gill and digestive gland of the mussel, Mytilus galloprovincialis. Animals were exposed (for 3, 6 and 12 h) to two sizes of silver nanoparticles (AgNP: <50 nm and <100 nm) prior to and after blockade of two endocytic pathways (amantadine blocks clathrin-mediated endocytosis while nystatin blocks caveolae-mediated endocytosis). Redox-proteomic tools were used to determine effects. Our results demonstrate the ability of both sizes of AgNP (<50 and <100 nm) to cause protein thiol oxidation and/or protein carbonylation. However, blockade of endocytotic routes mitigated AgNP toxicity. Differential ROS-related toxicity of AgNP to mussel tissues seemed to be linked to tissue-specific mode of action requirements. Cell uptake mechanism strongly influences toxicity of AgNPs in this filter-feeder.

Effect of exposure time, particle size and uptake pathways in immune cell lysosomal cytotoxicity of mussels exposed to silver nanoparticles.

Cytotoxicity evaluation of hemocytes (lysosomal membrane stability [LMS] assay) from Mytilus galloprovincialis Lamarck, exposed to a sublethal dose (100 µg/L) of two size of silver nanoparticles (AgNPs: <50 nm and <100 nm) - prior to and after inhibition of potential uptake pathways (i.e., clathrin- and caveolae-mediated endocytosis) within different times of exposure (3, 6, 12 h) - showed that there was a significant cytotoxic effect on immune cells of mussels exposed for different times to either AgNP size (p < 0.01); the greater effect was with the smaller size. However, hemocytes seemed more sensitive to the larger AgNP after clathrin-mediated endocytosis was blocked (p < 0.01); this was not so with inhibition of caveolae-mediated endocytosis. Dimethyl-sulfoxide (DMSO) did not impart a carrier-mediated effect despite an enhanced cytotoxicity when DMSO was present with AgNP. From these results, it is concluded that the immunotoxicity of AgNP in mussels was size-dependent as well as length of exposure-dependent. It was also clear that nanoparticles (NP) internalization mechanisms were a major factor underlying any toxicity.

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

BACKGROUND: Histopathological assessments approaches in bivalves have become an important tool in environmental toxicology. This study seeks to develop a quantitative histopathological index (Ih) and inflammation score as biomarkers in the aim to assess the health status of nanoparticles exposed mussels. METHODS: Digestive gland hematoxylin and eosin (H&E) stained sections from Mytilus galloprovincialis were assessed after in vivo exposure (for 3, 6 and 12 h) to silver nanoparticles (Ag-NPs < 50 nm and Ag-NPs < 100 nm) prior and after manipulating the potential uptake pathways (clathrin- and caveolae-mediated endocytosis) using amantadine and nystatin as blockers. Quantitative models evaluate the impacts of nanoparticles size, as well as their uptake routes within different time of exposure on the inflammation intensity, the digestive tubules histomorphometry and the histopathological indices. RESULTS: Silver nanoparticles clearly induced histopathological alterations in digestive gland (maximum inflammation 2.75 with AgNP < 100 nm [p < 0.05]; significant Ih with AgNP < 50 nm and AgNP < 100 nm at different time-points [p < 0.05]). Significant Ih were recorded after uptake routes were blockade: AgNP < 50 nm + nystatin and AgNP < 100 nm + amantadine; [p < 0.05] all time-points. CONCLUSIONS: Histopathological assessments showed to be promising tool in nanotoxicity which seems to depend on nanoparticles size, exposure time and interestingly to uptake routes. It was not clear: is it the length of exposure or the size of particles is more impactful.

Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis.

Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanoparticles (AgNP: <50 nm and <100 nm) on the frequency of hemocytes subpopulations as immunomodulation biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over different durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant variations in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that differing internalization mechanisms could be key factors impacting on the potential for NP in the environment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.

Histopathology and analyses of inflammation intensity in the gills of mussels exposed to silver nanoparticles: role of nanoparticle size, exposure time, and uptake pathways.

Environmentally induced perturbation of health parameters lead to morphological changes associated to the inflammatory response. Hematoxyline and eosin (H&E)-stained gill filaments sections were examined for such changes and inflammation intensity was scored according to a quantitative model in order to evaluate the health status of in vivo exposed (for 3, 6, and 12 h) mussels to silver nanoparticles (Ag-NPs <50 nm and Ag-NPs <100 nm) prior and after the inhibition of two potential uptake pathways (clathrin- and caveolae-mediated endocytosis) with the aid of pharmaceutical inhibitors (amantadine and nystatin). The impacts of the nanoparticles (NPs) size, as well as their uptake routes within different time of exposure on the inflammatory response were assessed. The results showed that Ag-NPs clearly induced morphological changes associated to the inflammatory response in gill tissues (Mann-Whitney p values were <.05). It is also clear that the length of the exposure as well as the NP size highly impacted inflammation intensity (highest histopathological indices recorded with Ag-NPs <100 nm). Also, the routes of NPs entry noticed to be major factor underlying inflammatory response (significant inflammation intensity reported with Ag-NPs <50 nm after blockade of uptake routes; p <.05). Throughout, it was concluded that inflammation intensity was related to NPs size and exposure time. Overall, uptake routes are shown to be the major factor underlying nanotoxicity.