[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA | International audience | The separate and combined in vitro toxic effects of antibiotics (ciprofloxacin, erythromycin, novobiocin, oxytetracycline, sulfamethazole and trimethoprim) commonly found in urban wastewater effluents were assessed on the immune parameters of Elliptio complanata at environmentally relevant concentrations. The observed responses were then compared to those produced by the physicochemical-treated wastewater effluent of a major city before and after the removal of microorganisms. Most of the selected antibiotics, separately and as mixture, induced changes in immune responses. The removal of microorganisms and fine particles from the effluent increased or decreased the resulting immunotoxic effects, depending of the observed parameter. The immunotoxic effects of erythromycin, sulfamethoxazole and trimethoprim were closely associated to the antibiotic mixture and the filtered effluent. In conclusion, the data revealed that the removal of fine particles and microorganisms from municipal effluents can alter the toxic nature of the effluent that is closely associated with the cumulative effects of antibiotics.

Tire particles pose a potential threat to terrestrial organisms because they are deposited in large quantities in the soil by tire wear abrasion, and moreover their chemical complexity poses an additional risk. Microplastics can affect several physiological processes in organisms, including those related to immunity. Therefore, we investigated the expression profile of selected immune-related genes (MnSod, Manganese Superoxide dismutase; Cat, Catalase; CypG, Cyclophilin G; Nos, Nitric oxide synthase; Ppae2a, Prophenoloxidase-activating enzyme 2a; Dscam, Down syndrome cell adhesion molecule; Myd88, Myeloid-differentiation factor 88; Toll4, Toll-like receptor 4; Mas-like, Masquerade-like protein) in haemocytes and the digestive gland hepatopancreas of terrestrial crustacean Porcellio scaber after two different time exposures (4 and 14 days) to tire particles in soil. Our results reveal for the first time the response of P. scaber after microplastic exposure at the transcriptome level. We observed time- and tissue-dependent changes in the expression of the analysed genes, with more pronounced alterations in haemocytes after 14 days of exposure. Some minor changes were also observed in hepatopancreas after 4 days. Changes in the expression profile of the analysed genes are a direct indication of a modulated immune status of the test organism, which, however, does not represent an adverse effect on the test organism under the given conditions. Nevertheless, the question remains whether the observed change in immune status affects the immunocompetence of the test organism.

The adverse effects of plastic waste and nanoplastics on the water environment have become a focus of global attention in recent years. In the present study, using adult Chinese mitten crabs (Eriocheir sinensis) as an animal model, the bioaccumulation and the in vivo and in vitro toxicity of polystyrene nanoplastics (PS NPs), alone or in combination with the bacteria, were investigated. This study aimed to investigate the effects of PS NPs on apoptosis and glucose metabolism in Chinese mitten crabs, and whether PS NPs could synergistically affect the antibacterial immunity of crabs. We observed that NPs were endocytosed by hemocytes, which are immune cells in crustaceans and are involved in innate immunity. The RNA sequencing data showed that after hemocytes endocytosed NPs, apoptosis and glucose metabolism-related gene expression was significantly induced, resulting in abnormal cell apoptosis and a glucose metabolism disorder. In addition, exposure to NPs resulted in changes in the antimicrobial immunity of crabs, including changes in antimicrobial peptide expression, survival, and bacterial clearance. In summary, NPs could be endocytosed by crab hemocytes, which adversely affected the cell apoptosis, glucose metabolism, and antibacterial immunity of Eriocheir sinensis. This study revealed the effects of NPs on crab immunity and lays the foundation for further exploration of the synergistic effect of NPs and bacteria.

Both microplastics and persistent organic pollutants (POPs) are ubiquitously present in natural water environment, posing a potential threat to aquatic organisms. While it has been suggested that the immune responses of aquatic organisms could be hampered by exposure to microplastics and POPs, the synergistic immunotoxic impact of these two types of pollutants remain poorly understood. In addition, little is known about the mechanism behind the immunotoxic effect of microplastics. Therefore, in the present study, the immunotoxicity of microplastics and two POPs, benzo[a]pyrene (B[a]P) and 17β-estradiol (E2), were investigated alone or in combination in a bivalve species, Tegillarca granosa. Evident immunotoxicity, as indicated by alterations of haemocyte count, blood cell composition, phagocytic activity, intracellular content of ROS, concentration of Ca²⁺ and lysozyme, and lysozyme activity, was revealed for both microplastics and the two POPs examined. In addition, the expression of six immune-, Ca²⁺ signalling-, and apoptosis-related genes was significantly altered by exposure of clams to the contaminants studied. Furthermore, the toxicity of POPs was generally aggravated by smaller microplastics (500 nm) and mitigated by larger ones (30 μm). This size dependent effect on POP toxicity may result from size dependent interactions between microplastics and POPs. Data obtained in this study also indicate that similar to exposure to B[a]P and E2, exposure to microplastics may hamper the immune responses of clams through a series of interdependent physiological and molecular processes.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 μM) with 0.4 μM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 μM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

Robust antimicrobial capability is crucial for marine organisms survival in complex ocean environments. Although the detrimental impacts of emergent pollutants on cellular immune response of marine bivalve mollusks were increasingly documented, the effects of bisphenol A (BPA) and microplastics (MPs) on humoral immune response and hemocyte chemotactic activity remain unclear. Therefore, in this study, the toxicities of BPA and MPs, alone or in combination, to the antimicrobial ability, humoral immune response, and hemocyte chemotactic activity were investigated in the blood clam Tegillarca granosa. Our data demonstrated that exposure of blood clams to BPA, MPs, and BPA-MPs for 2 weeks lead to significant reductions in their survival rates upon pathogenic bacterial challenge, indicating evident impairment of antimicrobial ability. Compared to control, the plasma of pollutant-incubated blood clams exhibited significantly less antimicrobial activity against the growth of V. harveyi, suggesting significant reduction in humoral immune effectors including defensin, lysozyme (LZM), and lectin. Moreover, hemocytes migration across the polycarbonate membrane to the serum containing chamber was markedly arrested by 2-week exposure to BPA, MPs, and BPA-MPs, suggesting a hampered chemotactic activity. In addition, the intracellular contents of ROS and protein carbonyl in hemocytes were markedly induced whereas the expression levels of key genes from the MAPK and actin cytoskeleton regulation pathways were significantly suppressed upon exposure. In this study, it was also found that BPA-MP coexposure was significantly more toxic than single exposures. In summary, our findings revealed that exposure to the pollutants tested possibly impair the antimicrobial ability of blood clam through (1) reducing the inhibitory effect of plasma on bacterial growth, the contents of humoral immune effectors, and the chemotactic activity of hemocytes, (2) interrupting IL-17 activation of MAPK signal pathway, (3) inducing intracellular ROS, elevating protein carbonylation levels, and disrupting actin cytoskeleton regulation in hemocytes.

Understanding the impacts of environmental pollutants on immune systems is indispensable in ecological and health risk assessments due to the significance of normal immunological functions in all living organisms. Bivalves as sentinel organisms with vital ecological importance are widely distributed in aquatic environments and their innate immune systems are the sensitive targets of environmental pollutants. As the central component of innate immunity, bivalve hemocytes are endowed with specialized endolysosomal systems for particle internalization and metal detoxification. These intrinsic biological features make them a unique cellular model for metal- and nano-immunotoxicology research. In this review, we firstly provided a general overview of bivalve's innate immunity and the classification and immune functions of hemocytes. We then summarized the recent progress on the interactions of metals and nanoparticles with bivalve hemocytes, with emphasis on the involvement of hemocytes in metal regulation and detoxification, the interactions of hemocytes and nanoparticles at eco/bio-nano interface and hemocyte-mediated immune responses to the exposure of metals and nanoparticles. Finally, we proposed the key knowledge gaps and future research priorities in deciphering the fundamental biological processes of the interactions of environmental pollutants with the innate immune system of bivalves as well as in developing bivalve hemocytes into a promising cellular model for nano-immuno-safety assessment.

Hemocytes are the main immune cells in bivalve mollusks and one of the sensitive targets for nanoparticle toxicity. Bivalve hemocytes consist of multiple functional heterogeneous cell types, but their different roles in immune system against foreign particles remain largely unknown. In order to clarify the different immune responses of hemocyte subpopulations to silver nanoparticles (AgNPs) and Ag ions, in this study, the Hong Kong oyster (Crassostrea hongkongensis) hemocytes were employed and separated into three subpopulations based on their cell size and granularity, including agranulocytes (R1), semigranulocytes (R2), and granulocytes (R3). We first demonstrated that AgNPs could rapidly enter into the oyster hemocytes within 3 h by phagocytosis process and resulted in different immune responses in hemocyte subpopulations. The most affected cell subtype by AgNPs was the granulocytes, followed by semigranulocytes, whereas agranulocytes were not affected following exposure to AgNPs. Interestingly, AgNPs induced the granule formation in semigranulocytes and further increased the proportion of granulocytes, whereas their ionic counterparts had no such effects on hemocyte composition, indicating the different detoxification mechanisms for nanoparticulate and ionic form. Following AgNP exposure, the dissolved Ag ions were accumulated in lysosomes and caused lysosomal dysfunction, indicating that lysosomes were the main targets for AgNP toxicity and the dissolved Ag ions were the main contributor of AgNP toxicity. Furthermore, AgNP exposure induced reactive oxygen production and impeded the lysosome function and phagocytosis in granulocytes, with impaired immunity system in oysters. Our study identified the different immune responses of oyster hemocyte subpopulations to AgNPs based on the in vitro short-term exposure assays, which may be applied to rapidly evaluate the ecotoxicological risks of different nanoparticles in aquatic systems.

The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies.