Toxicity of microplastics and triclosan, alone and in combination, to the fertilisation success of a broadcast spawning bivalve Tegillarca granosa.

Since most marine invertebrates adopted external fertilisation, their fertilisation process is particularly vulnerable to aquatic pollutants. Both antimicrobial ingredients and microplastics (MPs) are ubiquitous in aquatic environments; however, their synergistic effects on the fertilisation of marine invertebrates remain unclear. Therefore, in this study, the fertilisation toxicity of MPs and triclosan (TCS), alone and in combination, was investigated in the broadcast spawning bivalve Tegillarca granosa. Results showed that MPs and TCS significantly suppressed the fertilisation success of T. granosa. As the fertilisation success of broadcast spawning invertebrates depends on successful gamete collisions, gamete fusion, and egg activation, sperm swimming velocity, viability, gamete collision probability, ATP status, and ion-transport enzyme activities were also analysed to further ascertain the underlying toxicity mechanisms. In summary, our findings indicate that the presence of MPs may enhance the fertilisation toxicity of TCS by hampering sperm-egg collision probability, reducing gamete fusion efficiency, and restricting Ca2+ oscillation formation.

Circadian Rhythm and Neurotransmitters Are Potential Pathways through Which Ocean Acidification and Warming Affect the Metabolism of Thick-Shell Mussels.

Although the impacts of ocean acidification and warming on marine organisms have been increasingly documented, little is known about the affecting mechanism underpinning their interactive impacts on physiological processes such as metabolism. Therefore, the effects of these two stressors on metabolism were investigated in thick-shell mussel Mytilus coruscus in this study. In addition, because metabolism is primarily regulated by circadian rhythm and neurotransmitters, the impacts of acidification and warming on these two regulatory processes were also analyzed. The data obtained demonstrated that the metabolism of mussels (indicated by the clearance rate, oxygen consumption rate, ammonia excretion rate, O:N ratio, ATP content, activity of pyruvate kinase, and expression of metabolism-related genes) were significantly affected by acidification and warming, resulting in a shortage of energy supply (indicated by the in vivo content of ATP). In addition, exposure to acidification and warming led to evident disruption in circadian rhythm (indicated by the heartrate and the expression rhythm of Per2, Cry, and BMAL1) and neurotransmitters (indicated by the activity of acetyl cholinesterase and in vivo contents of ACh, GABA, and DA). These findings suggest that circadian rhythms and neurotransmitters might be potential routes through which acidification and warming interactively affect the metabolism of mussels.

Microplastics hamper the fertilization success of a broadcast spawning bivalve through reducing gamete collision and gamete fusion efficiency.

By employing external fertilization (broadcast spawning) as a mating strategy, the gametes and subsequent fertilization of various marine invertebrates are directly subjected to pollution. Although microplastics (MPs) are ubiquitous in marine environments, their potential effects on the fertilization of broadcast spawners remain largely unknown. Therefore in this study, the impacts of polystyrene MPs on the fertilization success of broadcast spawning bivalve (Tegillarca granosa) were investigated. In order to reveal the underlying mechanisms affecting fertilization, the sperm swimming performance, sperm ATP status, sperm viability, DNA integrity, gamete collision probability, gamete fusion efficiency, enzymatic antioxidants, and key ion transport enzyme activities were analyzed. The results showed that MPs weakened the sperm swimming performance through reducing ATP production and cell viability, thus leading to the decreased probability of gamete collision. Furthermore, MPs affected ion transport in the gametes by inducing oxidative stress, which resulted in gamete fusion failure. In conclusion, this study demonstrates that MPs could significantly decrease the fertilization success of T. granosa through reducing gamete collision and lowering gamete fusion efficiency.

Toxicities of three metal oxide nanoparticles to a marine microalga: Impacts on the motility and potential affecting mechanisms.

With the fast growth of the production and application of engineered nanomaterials (ENMs), nanoparticles (NPs) that escape into the environment have drawn increasing attention due to their ecotoxicological impacts. Motile microalgae are a type of primary producer in most ecosystems; however, the impacts of NPs on the motility of microalgae have not been studied yet. So the toxic impacts of three common metal oxide NPs (nTiO2, nZnO, and nFe2O3) on swimming speed and locomotion mode of a marine microalgae, Platymonas subcordiformis, were investigated in this study. Our results demonstrated that both the velocity and linearity (LIN) of swimming were significantly decreased after the exposure of P. subcordiformis to the tested NPs. In addition, the obtained data indicate that NPs may suppress the motility of P. subcordiformis by constraining the energy available for swimming, as indicated by the significantly lower amounts of intracellular ATP and photosynthetic pigments and the lower activities of enzymes catalyzing glycolysis. Incubation of P. subcordiformis with the tested NPs generally resulted in the overproduction of reactive oxygen species (ROS), aggravation of lipid peroxidation, and induction of antioxidant enzyme activities, suggesting that imposing oxidative stress, which may impair the structural basis for swimming (i.e. the membrane of flagella), could be another reason for the observed motility suppression. Moreover, NP exposure led to significant reductions in the cell viability of P. subcordiformis, which may be due to the disruption of the energy supply (i.e., photosynthesis) and ROS-induced cellular damage. Our results indicate that waterborne NPs may pose a great threat to motile microalgae and subsequently to the health and stability of the marine ecosystem.

Exposure to ethanol leads to midfacial hypoplasia in a zebrafish model of FASD via indirect interactions with the Shh pathway.

BACKGROUND: Gene-environment interactions are likely to underlie most human birth defects. The most common known environmental contributor to birth defects is prenatal alcohol exposure. Fetal alcohol spectrum disorders (FASD) describe the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD, but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem. RESULTS: We first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol-induced midfacial hypoplasia. We performed single-embryo RNA-seq during early embryonic stages to assess individual variation in the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol, we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly, but indirectly, interact to disrupt midfacial development. Ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor of the Wnt/PCP pathway, blebbistatin, phenocopies the effect of ethanol. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements. CONCLUSIONS: Our results show that the midfacial defects in ethanol-exposed vangl2 mutants are likely due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Ethanol exposure alters the position of a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.

The toxic impacts of microplastics (MPs) and polycyclic aromatic hydrocarbons (PAHs) on haematic parameters in a marine bivalve species and their potential mechanisms of action.

Microplastics (MPs) and polycyclic aromatic hydrocarbons (PAHs) are universally detected in the marine ecosystem and may exert adverse impacts on marine species. Although under realistic pollution scenarios, PAH pollution usually occurs as a mixture of different PAH compounds, the toxic impacts of PAH mixtures on marine organisms remain largely unknown to date, including their interactions with other emergent pollutants such as MPs. In this study, the single and combined toxic impacts of polystyrene MPs and a mixture of PAHs (standard mix of 16 representative PAHs) on haematic parameters were evaluated in the blood clam Tegillarca granosa. Our data demonstrated that blood clams treated with the pollutants examined led to decreased total haemocyte count (THC), changed haematic composition, and inhibited phagocytosis of haemocytes. Further analyses indicated that MPs and a mixture of PAHs may exert toxic impacts on haematic parameters by elevating the intracellular contents of reactive oxygen species (ROS), giving rise to lipid peroxidation (LPO) and DNA damage, reducing the viability of haemocytes, and disrupting important molecular signalling pathways (indicated by significantly altered expressions of key genes). In addition, compared to clams treated with a single type of pollutant, coexposure to MPs and a mixture of PAHs exerted more severe adverse impacts on all the parameters investigated, indicating a significant synergistic effect of MPs and PAHs.

Ocean acidification increases polyspermy of a broadcast spawning bivalve species by hampering membrane depolarization and cortical granule exocytosis.

Ensuring that oocytes are fertilized by a single sperm during broadcast spawning is crucial for the fertilization success of many marine invertebrates. Although the adverse impacts of ocean acidification (OA) on various marine species have been revealed in recent years, its impact on polyspermy and the underlying mechanisms involved remain largely unknown. Therefore, in the present study, the effect of OA on polyspermy risk was assessed in a broadcast spawning bivalve, Tegillarca granosa. In addition, the impacts of OA on the two polyspermy blocking processes, the fast block (membrane depolarization) and the permanent block (cortical reaction), were investigated. The results show that the exposure of oocytes to two future OA scenarios (pH 7.8 and pH 7.4) leads to significant increases in polyspermy risk, about 1.70 and 2.38 times higher than the control, respectively. The maximum change in the membrane potential during oocyte membrane depolarization markedly decreased to 15.79 % (pH 7.8) and 34.06 % (pH 7.4) of the control value. Moreover, the duration of oocyte membrane depolarization was significantly reduced to approximately 63.38 % (pH 7.8) and 21.91 % (pH 7.4) of the control. In addition, cortical granule exocytosis, as well as microfilament migration, were significantly arrested by OA treatment. Exposure to future OA scenarios also led to significant reductions in the ATP and Ca2+ content of the oocytes, which may explain the hampered polyspermy blocking. Overall, the present study suggests that OA may significantly increase polyspermy risk in T. granosa by inhibiting membrane depolarization and arresting cortical granule exocytosis.

Microplastics impair olfactory-mediated behaviors of goldfish Carassius auratus.

Recently, the ubiquitous presence of microplastics (MPs) has drawn worldwide concern over its potential threat to aquatic organisms. However, the effects of MPs on the olfactory ability of fish and the subsequent odorant evoked behaviors remain elusive. In the present study, we analyzed the potential olfactory toxicity of polystyrene (PS) MPs by assessing olfactory-driven behaviors of goldfish in response to odorants. Our results showed that the olfactory-driven behavioral responses of goldfish to L-cysteine and taurocholic acid were significantly hampered by a 28-day MP exposure. Further analysis demonstrated that exposure to MPs may suppress the expression of genes encoding olfactory G protein-coupled receptors, inhibit the enzyme activities of cation transport ATPases crucial for action potential generation, alter the in vivo contents of neurotransmitters as well as metabolites involved in the transduction of electrical signals, and cause olfactory bulb injury and neurotoxicity closely related to the processing of electrical signals. In conclusion, the results obtained in the present study suggest that MPs at environmentally relevant concentrations could impair the olfactory-mediated behavioral responses of goldfish, probably through hampering odorant identification, action potential generation, olfactory neural signal transduction, and olfactory information processing.

Fine polystyrene microplastics render immune responses more vulnerable to two veterinary antibiotics in a bivalve species.

Living in close proximity to the sediment of coastal areas, bivalves may be exposed to veterinary antibiotic residuals and microplastics (MPs) simultaneously. However, the immunotoxic impacts of veterinary antibiotics remain unknown in bivalves, let alone their interactions with MPs. Therefore, the immune responses of two representative veterinary antibiotics, oxytetracycline and florfenicol, was investigated in a bivalve species, the blood clam (Tegillarca granosa). The effects of the copresence of MPs on the immune responses triggered by these antibiotics were also analyzed. Results showed that exposure to antibiotics investigated led to significant alteration in hematic parameters and reduction in lectin content in serum. In addition to inducing ROS production, aggravating lipid peroxidation and DNA damage, and suppressing the hemocyte viability, antibiotic treatments also downregulated the expression of immune- and detoxification-related genes but upregulated apoptosis-related Caspase-3. Furthermore, the toxic impacts of antibiotics were found to be significantly increased by the copresence of MPs.

Modulatory function of calmodulin on phagocytosis and potential regulation mechanisms in the blood clam Tegillarca granosa.

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 Ca2+ 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 Ca2+-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 Ca2+ and downstream Ca2+-signaling-, actin cytoskeleton regulation-, and immune-related pathways offer candidate routes through which CaM modulates phagocytosis.

Immunotoxicity and neurotoxicity of bisphenol A and microplastics alone or in combination to a bivalve species, Tegillarca granosa.

Though invertebrates are one of the largest groups of animal species in the sea and exhibit robust immune and neural responses that are crucial for their health and survival, the potential immunotoxicity and neurotoxicity of the most produced chemical bisphenol A (BPA), especially in conjunction with microplastics (MPs), still remain poorly understood in marine invertebrate species. Therefore, the impacts of exposure to BPA and MPs alone or in combination on a series of immune and neural biomarkers were investigated in the invertebrate bivalve species blood clam (Tegillarca granosa). Evident immunotoxicity as indicated by alterations in hematic indexes was observed after two weeks of exposure to BPA and MPs at environmentally realistic concentrations. The expression of four immune-related genes from the NFκB signaling pathway was also found to be significantly suppressed by the BPA and MP treatment. In addition, exposure to BPA and MPs led to an increase in the in vivo contents of three key neurotransmitters (GABA, DA, and ACh) but a decrease in the expression of genes encoding modulatory enzymes and receptors for these neurotransmitters, implying the evident neurotoxicity of BPA and MPs to blood clam. Furthermore, the results demonstrated that the toxic impacts exerted by BPA were significantly aggravated by the co-presence of MPs, which may be due to interactions between BPA and MPs as well as those between MPs and clam individuals.

Immunotoxicity of petroleum hydrocarbons and microplastics alone or in combination to a bivalve species: Synergic impacts and potential toxication mechanisms.

Both the frequent occurrence of accidental petroleum spills and the ubiquitous presence of microplastics (MPs) in the sea may pose severe threats to marine species. However, the immunotoxic impacts of these two types of pollutants and the underlying toxication mechanisms still remain largely unknown in sessile filter-feeding bivalve mollusks. Therefore, the impacts of exposure to petroleum hydrocarbons and MPs alone or in combination on the total count, cell type composition, and phagocytic activity of hemocytes were investigated in the blood clam, Tegillarca granosa. In addition, the intracellular ROS content, cell viability, degree of DNA damage, and expression levels of genes from immune-, apoptosis-, and immunotoxicity-related pathways were analyzed to reveal the potential toxication mechanisms. The results demonstrated that exposure to petroleum hydrocarbons and MPs alone or in combination at environmentally realistic concentrations could exert significant immunotoxic impacts on the blood clam, which may be caused by alterations in a series of physiological and molecular processes. In addition, the immunotoxicity of petroleum hydrocarbons could be significantly aggravated by the copresence of MPs, which suggests that coexposure to these two pollutants deserves closer attention.

Microplastics Aggravate the Bioaccumulation of Two Waterborne Veterinary Antibiotics in an Edible Bivalve Species: Potential Mechanisms and Implications for Human Health.

Edible bivalves are one of the major types of seafood and may be subject to antibiotic and microplastics (MPs) coexposure under realistic scenarios. However, the effect of MPs on the bioaccumulation of antibiotics in edible bivalves and subsequent health risks for consumers remain poorly understood. Therefore, the bioaccumulation of two frequently detected veterinary antibiotics, oxytetracycline (OTC) and florfenicol (FLO), with or without the copresence of MPs was investigated in the blood clam. Health risks associated with the consumption of contaminated clams were also assessed. Furthermore, the activity of GST and expression of key detoxification genes were analyzed as well. The bioaccumulation of OTC and FLO in clams was found to be aggravated by MPs. Because the estimated target hazard quotients (THQs) were far less than the critical value, direct toxic effects of consuming contaminated clams are negligible. However, the dietary exposure doses of the human gut microbiota (DEGM) to the antibiotics tested were greater than or similar to corresponding minimum selective concentrations (MSC), indicating a potential antibiotic resistance risk. Moreover, the GST activity and expression of detoxification genes were significantly suppressed by MPs, suggesting that the disruption of detoxification represents one possible explanation for the aggravated bioaccumulation observed here.

Nanoparticles decrease the byssal attachment strength of the thick shell mussel Mytilus coruscus.

The extensive application of nanoparticles (NPs) drives their release into the ocean, which may pose a potential threat to marine organisms. Although the byssus is important for the survival of mussels, the effects of NPs on byssal attachment and the underlying molecular byssal responses remain largely unknown. Therefore, the impacts of three metal oxide NPs (nTiO2, nZnO, and nFe2O3) on the production and mechanical properties of byssal thread in the thick shell mussel M. coruscus were investigated in this study. The results showed that both mechanical properties (such as strength and extensibility) and morphology (diameter and volume) of byssal thread newly produced by M. coruscus were significantly affected after NP exposure, which resulted in an approximately 60-66% decrease in mussel byssal attachment strength. Downregulated expression of genes encoding mussel foot proteins, precursor collagen proteins, and proximal thread matrix proteins was also detected in this study, and this alteration may be one of the reasons for the weakened mechanical properties of byssal threads after NP exposure. This study indicated that NP pollution may hamper byssal attachment of M. coruscus and thereby pose a severe threat to the health of mussel individuals and the stability of the intertidal ecosystem.

Immunotoxicities of microplastics and sertraline, alone and in combination, to a bivalve species: size-dependent interaction and potential toxication mechanism.

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.

Acetylcholine suppresses phagocytosis via binding to muscarinic- and nicotinic-acetylcholine receptors and subsequently interfering Ca2+- and NFκB-signaling pathways in blood clam.

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 Ca2+. In addition, the expressions of genes from Ca2+ 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 Ca2+ homeostasis and subsequently interferes with downstream Ca2+ and NF-κB signaling pathways.