Hemolymph Serum Research Articles

First evidence of insecticidal potential of molecules produced during the parasitic phase of the endoparasitoid Cotesia flavipes

AbstractImmature endoparasitoids develop inside their hosts. Consequently, endoparasitoids need to circumvent the action of the immune system and reprogram the physiology of their hosts to develop successfully. For this, parasitoids release host regulation‐related molecules into their host’s hemolymph produced by the parasitoid itself or expressed in specific host cells infected by parasitoid symbiotic viruses. In addition to their roles in host regulation, these molecules may also present biotechnological potentials, such as for insecticidal purposes in crop protection. We performed a survey for the biotechnological potential of compounds present in the hemolymph serum with and without circulating cell contents of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) parasitized by Cotesia flavipes Cameron (Hymenoptera: Braconidae) as insecticides in feeding assays. Hemolymph serum from D. saccharalis larvae at 6 days after the start of the parasitism with and without circulating cell contents were collected and spread over artificial diet or over sugarcane leaves for feeding assays with D. saccharalis neonates. Hemolymph serum with or without circulating cell contents from non‐parasitized D. saccharalis larvae at the same age were used as controls. Subsequently, hemolymph serum plus circulating cell contents was fractioned using molecular filters and fractions with different molecular weight range were used for feeding assays with D. saccharalis neonates. Feeding larvae with leaf disks amended with hemolymph serum plus circulating cell contents from parasitized hosts resulted in an increase of ca. 30% in the mortality of D. saccharalis neonates. After fractioning hemolymph serum plus circulating cell contents, feeding assays with the >100 or 3–10 kDa fractions did not result in the previously observed increase in larval mortality, but resulted in a reduction of leaf consumption by neonates. Finally, we selected a parasitism‐specific protein band observed in the hemolymph >100 kDa fraction for mass spectrometry identification. This parasitism‐specific protein was similar to hymenopteran apolipophorins‐I. We conclude that there is an insecticidal potential in the hemolymph serum plus circulating cell contents of D. saccharalis larvae parasitized by C. flavipes and further investigations will be performed to identify, characterize, and uncover the biotechnological application of these molecules.

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.

Locust Hemolymph Conveys Erythropoietin-Like Cytoprotection via Activation of the Cytokine Receptor CRLF3.

The cytokine receptor-like factor 3 (CRLF3) is an evolutionary conserved class 1 cytokine receptor present in all major eumetazoan groups. Endogenous CRLF3 ligands have not been identified and the physiological responses mediated by mammalian CRLF3 are poorly characterized. Insect CRLF3 is activated by erythropoietin (Epo) and several related molecules that protect mammalian neurons from stress-induced apoptosis. However, insects neither express Epo nor “classical” Epo receptor. Cell-protective effects of insect hemolymph have been described for several species. In this study, we explored the possibility that the endogenous CRLF3 ligand is contained in locust hemolymph. PCR analyses confirmed expression of crfl3-transcripts in neurons and hemocytes of Locusta migratoria and Tribolium castaneum. Survival of locust hemocytes in primary cultures was significantly increased by supplementation of culture medium with locust hemolymph serum. Locust primary neuron cultures were also protected by locust hemolymph, though preceding exposure to fetal bovine serum changed the hemolymph dose-dependency of neuroprotection. Direct comparison of 10% hemolymph serum with recombinant human Epo in its optimal neuroprotective concentration revealed equivalent anti-apoptotic effects on hypoxia-exposed locust neurons. The same concentration of locust hemolymph serum also protected hypoxia-exposed T. castaneum neurons. This indicates that the neuroprotective factor in locust hemolymph is sufficiently conserved in insects to allow activation of neuroprotective receptors in different species. Locust hemolymph-induced neuroprotection in both L. migratoria and T. castaneum was abolished after RNAi-mediated suppression of crlf3-expression. In summary, we report the presence of a conserved endogenous cytokine in locust hemolymph that activates CRLF3 and connected anti-apoptotic processes in hemocytes and neurons. Identification and characterization of the CRLF3 ligand will promote knowledge about cytokine evolution and may unravel cell-protective agents with potential clinical application.

Functional and Morphological Changes Induced in Mytilus Hemocytes by Selected Nanoparticles.

Nanoparticles (NPs) show various properties depending on their composition, size, and surface coating, which shape their interactions with biological systems. In particular, NPs have been shown to interact with immune cells, that represent a sensitive surveillance system of external and internal stimuli. In this light, in vitro models represent useful tools for investigating nano-bio-interactions in immune cells of different organisms, including invertebrates. In this work, the effects of selected types of NPs with different core composition, size and functionalization (custom-made PVP-AuNP and commercial nanopolystyrenes PS-NH2 and PS-COOH) were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. The role of exposure medium was evaluated using either artificial seawater (ASW) or hemolymph serum (HS). Hemocyte morphology was investigated by scanning electron microscopy (SEM) and different functional parameters (lysosomal membrane stability, phagocytosis, and lysozyme release) were evaluated. The results show distinct morphological and functional changes induced in mussel hemocytes depending on the NP type and exposure medium. Mussel hemocytes may represent a powerful alternative in vitro model for a rapid pre-screening strategy for NPs, whose utilization will contribute to the understanding of the possible impact of environmental exposure to NPs in marine invertebrates.

ZnO, Ag and ZnO-Ag nanoparticles exhibit differential modes of toxic and oxidative action in hemocytes of mussel Mytilus galloprovincialis

The present study investigates the cytotoxic and oxidative effects of custom-made nanoparticles (NPs) on hemocytes of Mytilus galloprovincialis, utilizing hemolymph serum (HS) as exposure medium. Specifically, hemocyte lysosomal membrane destabilization (in terms of neutral red retention time assay/NRRT), superoxide anion (O2−), nitric oxide (NO, in terms of nitrites) and lipid peroxidation content (in terms of malondialdehyde/MDA equivalents) were determined in cells treated for 1 h with different concentrations (0.1–50 μg mL−1) of ZnO NPs, Ag NPs and ZnO-Ag NPs, as well as AgNO3 and/or ZnCl2 (bulk ions, respectively). According to the results, Ag NPs were more cytotoxic than ZnO-Ag NPs and/or ZnO NPs, while NRRT values observed in AgNO3 treated cells were lower than those of ZnCl2. Furthermore, high levels of both O2− and MDA were detected in cells treated with Ag NPs, ZnO-Ag NPs, and AgNO3 at concentrations lower than 5 μg mL−1, while high NO generation was observed only in cells treated with 5–25 μg mL−1 of ZnO NPs or ZnCl2. Despite the absence of data, regarding the formation of NP-serum protein corona complexes that could mediate NP surface energy and uptake efficiency, the current study firstly revealed that ZnO NPs, probably via their surface charge, particle agglomeration, and NP Zn+ release could promote an immune-related generation of O2− and NO via the respiratory burst stimulation, a process that is questioned in the case of Ag NPs and/or ZnO-Ag NPs. Moreover, ZnO-Ag NP interaction with biological membranes and their oxidative mode of action seemed to be regulated by the release and the antagonistic/synergistic response of its ionic counterparts (ZnO+ and Ag+), but further studies are needed to elucidate the oxidative mode of action of NP metal ions in complex NP mixtures.

Immunotoxicity of polystyrene nanoplastics in different hemocyte subpopulations of Mytilus galloprovincialis

Plastic represents 60-80% of litter in the ocean. Degradation of plastic to small fragments leads to the formation of microplastics (MPs <5 mm) and nanoplastics (NPs <1 µm). One of the most widely used and representative plastics found in the ocean is polystyrene (PS). Among marine organisms, the immune system of bivalves is recognized as suitable to assess nanomaterial toxicity. Hemocyte subpopulations [R1 (large granular cells), R2 (small semi-granular cells) and R3 (small agranular or hyaline cells)] of Mytilus galloprovincialis are specialized in particular tasks and functions. The authors propose to examine the effects of different sizes (50 nm, 100 nm and 1 μm) PS NPs on the different immune cells of mussels when they were exposed to (1 and 10 mg·L−1) of PS NPs. The most noteworthy results found in this work are: (i) 1 µm PS NPs provoked higher immunological responses with respect to 50 and 100 nm PS NPs, possibly related to the higher stability in size and shape in hemolymph serum, (ii) the R1 subpopulation was the most affected with respect to R2 and R3 concerning immunological responses and (iii) an increase in the release of toxic radicals, apoptotic signals, tracking of lysosomes and a decrease in phagocytic activity was found in R1.

Post-translational protein deimination signatures and extracellular vesicles (EVs) in the Atlantic horseshoe crab (Limulus polyphemus)

The horseshoe crab is a living fossil and a species of marine arthropod with unusual immune system properties which are also exploited commercially. Given its ancient status dating to the Ordovician period (450 million years ago), its standing in phylogeny and unusual immunological characteristics, the horseshoe crab may hold valuable information for comparative immunology studies.Peptidylarginine deiminases (PADs) are calcium dependent enzymes that are phylogenetically conserved and cause protein deimination via conversion of arginine to citrulline. This post-translational modification can lead to structural and functional protein changes contributing to protein moonlighting in health and disease. PAD-mediated regulation of extracellular vesicle (EV) release, a critical component of cellular communication, has furthermore been identified to be a phylogenetically conserved mechanism. PADs, protein deimination and EVs have hitherto not been studied in the horseshoe crab and were assessed in the current study. Horseshoe crab haemolymph serum-EVs were found to be a poly-dispersed population in the 20–400 nm size range, with the majority of EVs falling within 40–123 nm. Key immune proteins were identified to be post-translationally deiminated in horseshoe crab haemolymph serum, providing insights into protein moonlighting function of Limulus and phylogenetically conserved immune proteins. KEGG (Kyoto encyclopaedia of genes and genomes) and GO (gene ontology) enrichment analysis of deiminated proteins identified in Limulus revealed KEGG pathways relating to complement and coagulation pathways, Staphylococcus aureus infection, glycolysis/gluconeogenesis and carbon metabolism, while GO pathways of biological and molecular pathways related to a range of immune and metabolic functions, as well as developmental processes. The characterisation of EVs, and post-translational deimination signatures, revealed here in horseshoe crab, contributes to current understanding of protein moonlighting functions and EV-mediated communication in this ancient arthropod and throughout phylogeny.

In Vitro Effects of Hemolymph Serum of Potamon persicum Crab (HSPPC) on MCF-7 and MDA-231 Breast Cancer Cell Lines

Background: Breast cancer is the most common cause of cancer-related death in women worldwide. Therefore, there is an urget need to identify and develop therapeutic strategies against this deadly disease. This study is the first to investigate the effects of Hemolymph Serum of Potamon persicum Crab (HSPPC) on MCF-7 and MDA-231 breast cancer cell lines. Materials and Methods: LDH and MTT assays were performed on MCF-7 and MDA-231 breast cancer cell lines as well as human umbilical vein endothelial cells (HUVEC) to determine the cytotoxic and antiproliferative activity of the HSPPC at different concentrations. Further, the apoptosis inducing action of the hemolymph serum was determined by TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick end labeling) and cell death assay. Results: The IC50 values of HSPPC for MCF-7 and MDA-231 cell lines were 960±0.369 and 850±1.422 μg/mL, respectively. The growth of both MCF-7 and MDA-231 cell lines were significantly (P&lt;0.001) inhibited by HSPPC as compared with untreated controls at 48 hours. The results showed that HSPPC had no cytotoxic effects but significantly inhibited cell growth in a dose and time dependent manner. In addition, DNA fragmentation analysis (TUNEL) and cell death assay indicated induction of apoptosis by HSPPC in MCF-7 and MDA-231 cell lines. Conclusion: The results suggest that HSPPC contains bioactive compound(s) with potentials for the treatment of breast cancer.

Probiotic properties and immunomodulatory activity of gastrointestinal tract commensal bacterial strains isolated from the edible farmed snail Cornu aspersum maxima

The aim of this study was to determine the in vitro probiotic properties as well as the immunomodulatory activity of bacterial strains isolated from the gastrointestinal tract of the edible-farmed land snail Cornu aspersum maxima. Forty lactic acid bacterial strains (named Sgs1-40) were isolated from the intestinal tract and eight strains (named SgmA-H) from the oesophagus-crop of snails. Several criteria were used to examine whether they may be applied as snail-specific for the screening of the presumptive probiotic bacterial strains. Principal Component Analysis using criteria such as the tolerance of these strains to the pedal mucus, gastric mucus, gastric juices and low pH, as well as the expression of the cell surface traits of hydrophobicity, biofilm formation and autoaggregation capacity revealed discrimination of twelve strains exhibiting presumptive in vitro probiotic properties. Injection of eight of these strains, which were identified as Lactobacillus plantarum, in snail haemocoel increased the recruitment and phagocytic activity of amoebocytes in snail haemolymph. The Sgs14 and SgmB strains, exhibiting the highest immunostimulatory activity in haemolymph, were FITC-labelled and orally administrated to snails for ten days. The Sgs14 strain was able to adhere to intestinal mucosa of snails and stimulate the chemotactic and phagocytic activity of amoebocytes in haemolymph as well as the bactericidal activity of haemolymph serum. These responses are potentially mediated by the regulation of TLRs expression in the gut mucosa. These data indicate that the determination of properties such as snail mucus and gastric juice tolerance, cell surface traits for adhesion as well as increased chemotactic and phagocytic activity in snail haemolymph are eligible criteria to screen for snail-specific probiotics. To the best of our knowledge, this is the first work that investigates the probiotic properties of gastrointestinal microflora of the terrestrial farmed snail Cornu aspersum maxima.

In vivo immunomodulatory and antioxidant properties of nanoceria (nCeO2) in the marine mussel Mytilus galloprovincialis

Cerium nanoparticles (nCeO2) are increasingly utilized in a wide variety of industrial, environmental and biomedical applications, and are therefore expected to be released in the aquatic environment. Due to its peculiar redox properties, nCeO2 may present unique hazards to environmental and human health. Previous data showed that in the hemocytes of the marine bivalve Mytilus galloprovincialis, in vitro exposure to a particular type of nCeO2 (9 nm, characterized by negative ζ-potential, high H2O2 scavenging capacity and Ce3+/Ce4+ surface ratio) reduced basal ROS production, lysosomal membrane stability and phagocytic activity in the presence of hemolymph serum; the effects observed were partly ascribed to the formation of a SOD-protein corona in the hemolymph.In this work, the in vivo effects of this type of nCeO2 were investigated in mussels exposed to 100 μg/L nCeO2 for 96 h; several lysosomal, immune, inflammatory and antioxidant biomarkers were measured at cellular (hemocytes) and tissue (gills, digestive gland) level. Molecular responses were evaluated in hemocytes and digestive gland by determining expression of 11 selected genes related to known biological functions. The results show specific immunomodulatory and antioxidant effects of nCeO2 at different levels of biological organization in the absence of Cerium tissue accumulation. These data further support the redox mechanisms at the basis of the physiological effects of nCeO2. Finally, in order to evaluate the possible impact at the whole organism level, the effects of nCeO2 were evaluated in the 48 h embryotoxicity assay in a wide concentration range. However, nCeO2 exposure resulted in a small reduction in normal embryo development. Overall, the results demonstrate that in mussels nCeO2 can selectively modulate different physiological processes at different levels of biological organization.

Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development

Silver nanoparticles (AgNP), one of the main nanomaterials for production and use, are expected to reach the aquatic environment, representing a potential threat to aquatic organisms. In this study, the effects of bare AgNPs (47 nm) on the marine mussel Mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. The effects were compared with those of ionic Ag+(AgNO3). In vitro short-term exposure (30 min) of hemocytes to AgNPs induced small lysosomal membrane destabilization (LMS EC50 = 273.1 μg/mL) and did not affect other immune parameters (phagocytosis and ROS production). Responses were little affected by hemolymph serum (HS) as exposure medium in comparison to ASW. However, AgNPs significantly affected mitochondrial membrane potential and actin cytoskeleton at lower concentrations. AgNO3 showed much higher toxicity, with an EC50 = 1.23 μg/mL for LMS, decreased phagocytosis and induced mitochondrial and cytoskeletal damage at similar concentrations.Both AgNPs and AgNO3 significantly affected Mytilus embryo development, with EC50 = 23.7 and 1 μg/L, respectively. AgNPs caused malformations and developmental delay, but no mortality, whereas AgNO3 mainly induced shell malformations followed by developmental arrest or death.Overall, the results indicate little toxicity of AgNPs compared with AgNO3; moreover, the mechanisms of action of AgNP appeared to be distinct from those of Ag+. The results indicate little contribution of released Ag+ in our experimental conditions. These data provide a further insight into potential impact of AgNPs in marine invertebrates.

Cytotoxicity of CeO2 nanoparticles using in vitro assay with Mytilus galloprovincialis hemocytes: Relevance of zeta potential, shape and biocorona formation

Over the last decades, the growth in nanotechnology has provoked an increase in the number of its applications and consumer products that incorporate nanomaterials in their formulation. Metal nanoparticles are released to the marine environment and they can interact with cells by colloids forces establish a nano-bio interface. This interface can be compatible or generate bioadverse effects to cells. The daily use of CeO2 nanoparticles (CeO2 NPs) in industrial catalysis, sunscreen, fuel cells, fuel additives and biomedicine and their potential release into aquatic environments has turned them into a new emerging pollutant of concern. It is necessary to assess of effects of CeO2 NPs in aquatic organisms and understand the potential mechanisms of action of CeO2 NP toxicity to improve our knowledge about the intrinsic and extrinsic characteristic of CeO2 NPs and the interaction of CeO2 NPs with biomolecules in different environment and biological fluids. The conserved innate immune system of bivalves represents a useful tool for studying immunoregulatory responses when cells are exposed to NPs. In this context, the effects of two different CeO2 NPs with different physico-chemical characteristics (size, shape, zeta potential and Ce+3/Ce+4 ratio) and different behavior with biomolecules in plasma fluid were studied in a series of in vitro assays using primary hemocytes from Mytilus galloprovincialis. Different cellular responses such as lysosome membrane stability, phagocytosis capacity and extracellular reactive oxygen species (ROS) production were evaluated. Our results indicate that the agglomeration state of CeO2 NPs in the exposure media did not appear to have a substantial role in particle effects, while differences in shape, zeta potential and biocorona formation in NPs appear to be important in provoking negative impacts on hemocytes.The negative charge and the rounded shape of CeO2 NPs, which formed Cu, Zn-SOD biocorona in hemolymph serum (HS), triggered higher changes in the biomarker of stress (LMS) and immunological parameters (ROS and phagocytosis capacity). On the other hand, the almost neutral surface charge and well-faceted shape of CeO2 NPs did not show either biocorona formation in HS under tested conditions or significant responses. According to the results, the most relevant conclusion of this work is that not only the physicochemical characterization of CeO2 NPs plays an important role in NPs toxicity but also the study of the interaction of NPs with biological fluids is essential to know it behavior and toxicity at cellular level.

Evaluation of Beauveria bassiana infection in thehemolymph serum proteins of the housefly, Musca domestica L. (Diptera: Muscidae).

Beauveria bassiana plays a prominent role in biocontrol of houseflies, Musca domestica (L.). Thus, a deeper insight into immune response of M. domestica during B. bassiana infection was warranted to assist the production of more efficient mycoinsecticides. The present study investigates changes in protein profile of M. domestica hemolymph serum post B. bassiana infection using two-dimensional difference gel electrophoresis (2D-DIGE) followed by identification of selected proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The non-infected or control group of flies showed an expression of 54 proteins, while M. domestica infected with B. bassiana expressed a total of 68 hemolymph serum proteins. Thirty three proteins were expressed in both groups of houseflies, whereas 35 proteins were exclusively expressed in infected flies and 21 proteins were exclusively expressed in control flies. Among the 33 proteins which were expressed in both groups of houseflies, 17 proteins showed downregulation, while16 proteins were upregulated in the infected flies compared to the non-infected ones. The results from this study are expected to facilitate better understanding of insect's immune response mechanism.

Photocatalytic Fe-doped n-TiO 2 : From synthesis to utilization of in vitro cell models for screening human and environmental nanosafety

The utilization of different types nanomaterials (NMs) in environmental remediation and wastewater treatment requires information on the potential harmful effects on human and environmental health. In this light, the utilization of human cell models together with cells from lower organisms, representative of different environmental compartments, could represent a valuable tool for the in vitro screening of the potential toxicity of different NMs used in nanoremediation. Among NMs, n-TiO2, because of its peculiar optical and chemical properties, is widely applied for photosensitized UV oxidation of organic pollutants. Moreover, development in design of metal- and non metal- doped TiO2 with extended photocatalytic activity in the visible region represents the subject of ongoing research. In this work, the cytotoxic effects of three different types of recently synthetized Fe-doped n-TiO2 were compared in two cell models widely utilized for screening cellular toxicity of NMs in humans and aquatic organisms, human vascular endothelial cells (HECV) and immune cells (hemocytes) of the marine invertebrate, the mussel Mytilus spp, respectively. Parallel studies were carried out using N-doped n-TiO2. The results indicate both distinct and common behavior (agglomeration state) in different media (human cell culture medium and mussel hemolymph serum) and biological effects (cytotoxicity, nitric oxide production) of different types of doped- n-TiO2 in different cell models. Although in vitro studies represent a first step in the toxicological assessment of NMs, studies comparing their effects on human and aquatic invertebrate cells that take into account the effects of different exposure media represent an useful tool for evaluating potential cytotoxicity of those NMs, like TiO2-based photocatalytic NMs, widely applied in environmental remediation, and whose potential risks are poorly understood.

Comparison of Methods for Determining Nutritional Condition in Spiny Lobsters

ABSTRACT Four previously published metrics for determining nutritional condition in decapods were evaluated for efficacy, specifically a serum protein index and three hepatopancreas-based indices (dry weight index, percent tissue dry weight, and relative weight). Using the Caribbean spiny lobster Panulirus argus (Latreille, 1804) as a model, lobsters were subjected to 5 wk of controlled diets in a laboratory-feeding trial. Three of the four indices detected significant differences between lobsters fed 15% or 100% of their daily maximum food intake, but lobster size and molt stage significantly influenced several metrics. Dry weight index of the hepatopancreas performed best, but relative weight of the digestive gland was the least affected by lobster size and molt stage. As both are destructive methods that require dissection of the lobster, these may not be useful for all studies. Hemolymph serum protein concentration is nondestructive and can be assessed rapidly in the laboratory or field, but it is str...

Photocatalytic Fe-doped n-TiO 2 : From synthesis to utilization of in vitro cell models for screening human and environmental nanosafety

The utilization of different types nanomaterials (NMs) in environmental remediation and wastewatertreatment requires information on the potential harmful effects on human and environmental health. Inthis light, the utilization of human cell models together with cells from lower organisms, representativeof different environmental compartments, could represent a valuable tool for the in vitro screening of thepotential toxicity of different NMs used in nanoremediation. Among NMs, n-TiO 2 , because of its peculiaroptical and chemical properties, is widely applied for photosensitized UV oxidation of organic pollutants.Moreover, development in design of metal- and non metal- doped TiO 2 with extended photocatalyticactivity in the visible region represents the subject of ongoing research.In this work, the cytotoxic effects of three different types of recently synthetized Fe-doped n-TiO 2were compared in two cell models widely utilized for screening cellular toxicity of NMs in humans andaquatic organisms, human vascular endothelial cells (HECV) and immune cells (hemocytes) of the marineinvertebrate, the mussel Mytilus spp , respectively. Parallel studies were carried out using N-doped n-TiO 2 .The results indicate both distinct and common behavior (agglomeration state) in different media (humancell culture medium and mussel hemolymph serum) and biological effects (cytotoxicity, nitric oxide production)of different types of doped- n-TiO 2 in different cell models. Although in vitro studies represent a first step in the toxicological assessment of NMs, studies comparing their effects on human and aquatic invertebrate cells that take into account the effects of different exposure media represent an useful tool for evaluating potential cytotoxicity of those NMs, like TiO 2 -based photocatalytic NMs, widely applied in environmental remediation, and whose potential risks are poorly understood.

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.

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.

Haemolymph from Mytilus galloprovincialis: Response to copper and temperature challenges studied by 1H-NMR metabonomics

Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16°C) or high temperature (24°C) and in the absence or presence (5μg/L, 20μg/L, or 40μg/L) of sublethal copper (Cu2+). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the 1H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16°C became detectable at a dose of 20μg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24°C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.

Temporal Variation in Immune Components of the White Grub Phyllophaga polyphylla (Bates) (Coleoptera: Melolonthidae).

Ecological immunology assumes that the host immune efficiency is correlated with its survival after pathogen challenge. To test this hypothesis, we challenged Phyllophaga polyphylla (Bates) larvae with the naturally occurring fungus Metarhizium pingshaense on two consecutive years (2011 and 2012). In each year, we injected the blastospores of M. pingshaense and then used levels of prophenoloxidase (proPO), phenoloxidase (PO) and total haemolymph serum protein as indicators of immune efficiency. Larvae were injected with (1) phosphate buffered saline (PBS) + Tween and viable blastospores of M. pingshaense, (2) PBS + Tween and non-viable blastospores of M. pingshaense, (3) PBS + Tween, or (4) non-manipulated. Overall, levels of PO, proPO and total haemolymph serum protein in larvae after 12h were similar amongst treatments within each year of collection. However, larvae collected in 2011 showed higher PO and proPO activity but lower total haemolymph serum protein compared with larvae collected in 2012. A survival study injecting viable blastospores showed that larvae collected in both years died within 48h; however, when non-viable blastospores were injected, which were still toxic to larvae, mortality was greater in larvae collected in 2011 compared with larvae collected in 2012. Altogether, these results indicate that PO, proPO and total haemolymph serum protein do not predict immune strength of P. polyphylla against blastospores of M. pingshaense, but higher values of PO and proPO were correlated with higher survival rates against non-infective but toxic agents. The possible role of some abiotic factors over the differences observed for immune components of P. polyphylla in different years of collection is discussed.