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Unlike vertebrate species, invertebrates lack antigen-antibody mediated immune response and mainly rely on haemocyte phagocytosis to fight against pathogen infection. Recently, studies conducted in model vertebrates demonstrated that the multifunctional protein calmodulin (CaM) plays an important role in regulating immune responses. However, the intrinsic relation between CaM and phagocytosis process remains poorly understood in invertebrate species such as bivalve mollusks. Therefore, in the present study, the immunomodulatory function of CaM on haemocyte phagocytosis was verified in the blood clam, Tegillarca granosa, using the CaM-specific inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Results obtained show that CaM inhibition significantly suppressed the phagocytic activity of haemocytes. In addition, CaM inhibition constrained intracellular Ca 2+ elevation, hampered actin cytoskeleton assembly, suppressed calcineurin (CaN) activity, and disrupted NF-κB activation in haemocytes upon LPS induction. Furthermore, expression of seven selected genes from the actin cytoskeleton regulation- and immune-related pathways were significantly downregulated whereas those of CaM and CaN from the Ca 2+ -signaling pathway were significantly upregulated by in vitro incubation of haemocytes with W-7. For the first time, the present study demonstrated that CaM play an important role in phagocytosis modulation in bivalve species. In addition, the intracellular Ca 2+ and downstream Ca 2+ -signaling-, actin cytoskeleton regulation-, and immune-related pathways offer candidate routes through which CaM modulates phagocytosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Though immunomodulation via cholinergic neurotransmitter acetylcholine (ACh), an important part of neuroendocrine-immune (NEI) regulatory network, has been well established in vertebrate species, the mechanisms remain poorly understood in invertebrates. In the present study, the immunomodulatory effect of ACh on haemocyte phagocytosis was investigated in an invertebrate bivalve species, Tegillarca granosa. Data obtained showed that in vitro ACh incubation suppressed phagocytic activity of haemocytes along with a significant elevation in intracellular Ca 2+ . In addition, the expressions of genes from Ca 2+ signaling pathway were significantly induced whereas those from NF-κB signaling pathway were significantly down-regulated by ACh incubation. Furthermore, these adverse impacts of ACh were significantly relieved by the blocking of muscarinic acetylcholine receptors (mAChRs) or nicotinic acetylcholine receptors (nAChRs) using corresponding antagonists. Our study suggests that ACh suppresses phagocytosis via binding to both mAChRs and nAChRs, which disrupts intracellular Ca 2+ homeostasis and subsequently interferes with downstream Ca 2+ and NF-κB signaling pathways., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Phagocytosis suppression induced by nanoparticles (NPs) exposure is increasingly reported in marine species. However, the mechanisms underlying this impact remain poorly understood. In order to improve our present understanding of the immunotoxicity of NPs, acute (96 h) TiO 2 NP exposure and rescue trials via exogenous supply of Ca 2+ were performed in the blood clam, Tegillarca granosa. The results show that the phagocytosis rate, cell viability, and intracellular Ca 2+ concentration of haemocytes were significantly suppressed, whereas the intracellular ROS concentration of haemocytes significantly increased upon nTiO 2 exposure. Exposure to nTiO 2 also led to the significant downregulation of Caspase-3, Caspase-6, apoptosis regulator Bcl-2, Bcl-2-associated X, calmodulin kinase II, and calmodulin kinase kinase II. Furthermore, the toxic impacts of nTiO 2 were partially mitigated by the addition of exogenous Ca 2+ , as indicated by the recovery tendency in almost all the measured parameters. The present study indicates that Ca 2+ signaling could be one of the key pathways through which nTiO 2 attacks phagocytosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Phagocytosis suppression induced by nanoparticles (NPs) exposure is increasingly reported in marine species. However, the mechanisms underlying this impact remain poorly understood. In order to improve our present understanding of the immunotoxicity of NPs, acute (96 h) TiO 2 NP exposure and rescue trials via exogenous supply of Ca2+ were performed in the blood clam, Tegillarca granosa. The results show that the phagocytosis rate, cell viability, and intracellular Ca2+ concentration of haemocytes were significantly suppressed, whereas the intracellular ROS concentration of haemocytes significantly increased upon nTiO 2 exposure. Exposure to nTiO 2 also led to the significant downregulation of Caspase-3, Caspase-6, apoptosis regulator Bcl-2, Bcl-2-associated X, calmodulin kinase II, and calmodulin kinase kinase II. Furthermore, the toxic impacts of nTiO 2 were partially mitigated by the addition of exogenous Ca2+, as indicated by the recovery tendency in almost all the measured parameters. The present study indicates that Ca2+ signaling could be one of the key pathways through which nTiO 2 attacks phagocytosis. Image 1051475 • nTiO 2 suppressed phagocytosis, cell viability, and Ca2+ content of haemocytes. • The ROS concentration in haemocytes was induced by nTiO 2 exposure. • nTiO 2 downregulated the expression of apoptosis and Ca2+ signaling related genes. • The toxic impacts of nTiO 2 were partially mitigated by adding exogenous Ca2+ The toxic effects of nTiO 2 on phagocytosis, cell viability, and apoptosis of Tegillarca granosa and the rescue effects of exogenous Ca2+ were demonstrated. [ABSTRACT FROM AUTHOR]

• Exposure blood clams to four NPs led to reduced THC, altered cell composition, and constrained phagocytosis of haemocytes. • The intracellular contents of ROS and the degree of DNA damage of haemocytes were significantly induced. • The haemocyte viability was suppressed upon NP exposures. • Exposure of blood clams to tested NPs led to significant increases in the in vivo contents of neurotransmitters. • NP exposures suppressed the expression of the immune- and neurotransmitter-related genes tested. The increasing application of nanomaterials drives the unintentional release of nanoparticles (NPs) into the ocean, which may pose a potential threat to marine organisms. It has been demonstrated that exposure to NPs could chanllenge the immune responses of marine species. However, the affecting mechanism behind remains poorly understood. In this study, the immunotoxic impacts and the mechanisms underpinning the effects of four major NPs, including nZnO, nFe 2 O 3 , nCuO, and carbon nanotube (MWCNT), were investigated in blood clam, Tegillarca granosa. The results showed that exposure to tested NPs resulted in reduced total counts, altered cell composition, and constrained phagocytic activities of haemocytes. The intracellular contents of reactive oxygen species (ROS) and the degree of DNA damage of haemocytes were significantly induced, whereas the haemocyte viability was suppressed. Furthermore, NP exposures led to significant increases in the in vivo contents of neurotransmitters. Down-regulations of the immune- and neurotransmitter-related genes were detected as well. Our data suggest that NP exposures hampered the immune responses of blood clams most likely through (1) inducing ROS, causing DNA damage, and reducing cell viability of haemocytes, (2) altering the in vivo contents of neurotransmitters, and (3) affecting the expression of immune- and neurotransmitter-related genes. [ABSTRACT FROM AUTHOR]

Exposure to heavy metals such as Cadmium (Cd) may exert detrimental impacts on the immune responses of marine bivalve species. However, the immunotoxicity of Cd on blood clams remains unknown to date. Furthermore, though Cd 2+ is known to compete with calcium (Ca 2+ ) ions for their binding sites in cells and inhibit Ca 2+ influx, whether Cd 2+ weakens the immune responses of marine bivalves through inducing intracellular Ca 2+ disorders still remains unclear. Therefore, the immunotoxicity of Cd 2+ at different waterborne Ca 2+ concentrations on blood clam, Tegillarca granosa , were investigated in the present study. Results obtained demonstrated that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were all significantly reduced after 10 days exposure of individuals to 25 μg/L Cd 2+ . However, when the waterborne Ca 2+ concentrations were elevated by 10% and 20% (approximately 370 and 410 mg/L, respectively), mitigation effects on the immune responses of individuals were detected. In addition, though the expressions of genes from the Ca 2+ signaling and Ca 2+ -related apoptosis pathways were significantly altered by Cd 2+ exposure, the expression patterns of these genes were similar to that of the control when the waterborne Ca 2+ concentrations were elevated, suggesting a relieving effect of waterborne Ca 2+ on Cd 2+ induced toxicity to haemocytes. The results obtained in the present study revealed that waterborne Cd 2+ may hamper the immune responses of T. granosa through influencing Ca 2+ signaling and Ca 2+ -related apoptosis pathways, which can be partially mitigated by elevating the waterborne Ca 2+ concentrations. [ABSTRACT FROM AUTHOR]

Persistent organic pollutants (POPs) are known to converge into the ocean and accumulate in the sediment, posing great threats to marine organisms such as the sessile bottom burrowing bivalves. However, the immune toxicity of POPs, such as B[a]P, under future ocean acidification scenarios remains poorly understood to date. Therefore, in the present study, the impacts of B[a]P exposure on the immune responses of a bivalve species, Tegillarca granosa , under present and future ocean acidification scenarios were investigated. Results obtained revealed an increased immune toxicity of B[a]P under future ocean acidification scenarios in terms of reduced THC, altered haemocyte composition, and hampered phagocytosis, which may attribute to the synergetic effects of B[a]P and ocean acidification. In addition, the gene expressions of pathogen pattern recognition receptors (TLR1, TLR2, TLR4, TLR6), pathway mediators (TRAF6, TAK1, TAB2, IKKα and Myd88), and effectors (NF-ĸB) of the important immune related pathways were significantly down-regulated upon exposure to B[a]P under future ocean acidification scenarios. Results of the present study suggested an increased immune toxicity of B[a]P under future ocean acidification scenarios, which will significantly hamper the immune responses of T. granosa and subsequently render individuals more susceptible to pathogens challenges. [ABSTRACT FROM AUTHOR]

Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABA A and GABA B receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl−, which was shown to be leveled down to normal by blocking the ionotropic GABA A receptor. Treatment with GABA A receptor antagonist also rescued the suppression of GABA A receptor-associated protein (GABARAP), KCC , TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), and nuclear factor kappa B subunit 1 (NFκB) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6 , IKKα , and NFκB. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABA B receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABA A and GABA B receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates. • GABA incubation significantly constrained hemocyte phagocytosis in blood clam. • GABA-induced immunosuppression was relieved by blocking of GABA A and GABA B receptors. • Cl−-NFκB could be downstream pathway of the binding of GABA and GABA A receptor. • The binding of GABA to GABA B receptor triggered cAMP-NFκB and Ca2+-NFκB pathways. [ABSTRACT FROM AUTHOR]

• MPs and Ser alone or in combination suppressed the immune responses of T. granosa. • An evident synergistic immuno-toxic effect of Ser and nanoscale MPs was observed. • MPs could result in aggravated toxicity due to the hampered detoxification of Ser. • Ser-MPs coexposure significantly altered gene expression profile in digestive glands. Although coexposure to pharmaceuticals and microplastics (MPs) may frequently occur, the synergistic impact of MPs and antidepressants on marine species still remains poorly understood. In this study, the immunotoxicities of polystyrene MPs (diameters 500 nm and 30 μm) and sertraline (Ser), alone and in combination, were investigated in a bivalve mollusk Tegillarca granosa. Results showed that both MPs and Ser significantly suppressed the immune responses of T. granosa. In addition, though the toxic effect of Ser was not affected by microscale MPs, an evident synergistic immuno-toxic effect was observed between Ser and nanoscale MPs, which indicates a size-dependent interaction between the two. To further ascertain the underlying toxication mechanisms, the intracellular content of reactive oxygen species, apoptosis status, ATP content, pyruvate kinase activity, plasma cortisol level, and in vivo concentrations of neurotransmitters and cytochrome P450 1A1 were analysed. A transcriptomic analysis was also performed to reveal global molecular alterations following Ser and/or MPs exposure. The obtained results indicated that the presence of nanoscale MPs may enhance the immunotoxicity of Ser by (i) inducing apoptosis of haemocytes and, hence, reducing the THC; (ii) constraining the energy availability for phagocytosis; and (iii) hampering the detoxification of Ser. [ABSTRACT FROM AUTHOR]