Exposure to nanopollutants (nZnO) enhances the negative effects of hypoxia and delays recovery of the mussels' immune system.

Aquatic environments face escalating challenges from multiple stressors like hypoxia and nanoparticle exposure, with impact of these combined stressors on mussel immunity being poorly understood. We investigated the individual and combined effects of short-term and long-term hypoxia and exposure to zinc oxide nanoparticles (nZnO) on immune system of the mussels (Mytilus edulis). Hemocyte functional traits (mortality, adhesion capacity, phagocytosis, lysosomal abundance, and oxidative burst), and transcript levels of immune-related genes involved in pathogen recognition (the Toll-like receptors, the complement system components, and the adaptor proteins MyD88) were assessed. Short-term hypoxia minimally affected hemocyte parameters, while prolonged exposure led to immunosuppression, impacting hemocyte abundance, viability, phagocytosis, and defensin gene expression. Under normoxia, nZnO stimulated immune responses of mussel hemocytes. However, combined nZnO and hypoxia induced more pronounced and rapid immunosuppression than hypoxia alone, indicating a synergistic interaction. nZnO exposure hindered immune parameter recovery during post-hypoxic reoxygenation, suggesting persistent impact. Opposing trends were observed in pathogen-sensing and pathogen-elimination mechanisms, with a positive correlation between pathogen-recognition system activation and hemocyte mortality. These findings underscore a complex relationship and potential conflict between pathogen-recognition ability, immune function, and cell survival in mussel hemocytes under hypoxia and nanopollutant stress, and emphasize the importance of considering multiple stressors in assessing the vulnerability and adaptability of mussel immune system under complex environmental conditions of anthropogenically modified coastal ecosystems.

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants.

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

Sonochemical regulation of oxygen vacancies for Bi2WO6 nanosheet-based photoanodes to promote photoelectrochemical performance.

Integration of oxygen vacancies (Vo) into nanostructured semiconductor-based photocatalysts has been recognized as a promising strategy for enhancing the performance of photoelectrochemical (PEC) water splitting. However, precisely controlling the Vo concentration in photocatalysts via an effective and tunable approach remains challenging. Herein, a series of optimized bismuth tungstate (Bi2WO6) nanosheet-based photoanodes with varying concentrations of Vo were prepared by a sonochemical method with in situ cavitation detection, which enables accurate manipulation of the acoustic cavitation intensity applied to the surface of Bi2WO6 photoanodes in alkaline solution. Based on the analysis of the Vo concentration and sound field characteristics, the mechanism of sonochemical regulation of Vo in Bi2WO6 nanosheets was interpreted. Specifically, the increase in Vo concentration can be attributed to the enhancement of Bi-O bond dissociation. This enhancement is influenced not only by the intensified impact of shear force and the generation of active radicals by transient cavitation, but also by the accelerated diffusion of the reactant, a result of stable cavitation. By optimizing the transient and stable cavitation intensity, a Vo-rich Bi2WO6 photoanode was obtained without altering the microstructure of Bi2WO6 nanosheets. The presence of high concentration Vo facilitates the interfacial chemical reactivity and the transmission of photogenerated carriers, leading to the drastic promotion of the PEC water splitting performance. The transient photocurrent density of the Vo-rich Bi2WO6 photoanode reaches 69.2 µA cm-2 (1.23 V vs. RHE), 7.86 times that of the untreated Bi2WO6 photoanode. Additionally, the charge injection efficiency increases to 35.4%. This work provides a controllable and effective method for defect engineering of nanostructured semiconductor-based electrodes.

Incidence and risk outcomes of second primary malignancy of patients with post-operative colorectal cancer.

BACKGROUND AND AIMS: This study aimed to investigate the incidence and the risk factors of incidence for second primary malignancies (SPMs) onset among survivors diagnosed with colorectal cancer (CRC). METHODS: A large population-based cohort study was performed. Data of patients diagnosed with CRC was identified and extracted from 8 cancer registries of Surveillance, Epidemiology, and End Results database from January 1990 to December 2017. The outcome of interest was percentage and common sites of SPM onset after primary CRC diagnosis. The cumulative incidence and standardize incidence rates (SIRs) were also reported. Afterwards, we estimated sub-distribution hazards ratios (SHRs) and relative risks (RRs) for SPM occurrence using multivariable competing-risk and Poisson regression models, respectively. RESULTS: A total of 152,402 patients with CRC were included to analyze. Overall, 23,816 patients of all CRC survivors (15.6%) were reported SPM occurrence. The highest proportion of SPMs development after primary CRC diagnosis was second CRC, followed by lung and bronchus cancer among all survivors. Also, CRC survivors were more susceptible to develop second gastrointestinal cancers (GICs). Besides, pelvic cancers were analyzed with a relative high proportion among patients who received RT in comparison to those without RT. The cumulative incidence of all SPMs onset was 22.16% (95% CI: 21.82-22.49%) after near 30-year follow-up. Several factors including older age, male, married status, and localized stage of CRC were related to the high risk of SPMs onset. In treatment-specific analyses, RT was related to a higher cumulative incidence of SPMs occurrence (all SPMs: 14.08% vs. 8.72%; GICs: 2.67% vs. 2.04%; CRC: 1.01% vs. 1.57%; all p < 0.01). Furthermore, the increased risk of SPMs onset was found among patients who received RT than patients within the NRT group (SHR: 1.50, 95% CI: 1.32-1.71), p < 0.01; RR: 1.61, 95% CI: 1.45-1.79, p < 0.01). CONCLUSION: The present study described the incidence pattern of SPM among CRC survivors and identified the risk factors of the SPM onset. RT treatment for patients diagnosed with CRC may increase the risk of SPMs occurrence. The findings suggest the need for long-term follow-up surveillance for these patients.

Genome-wide identification and validation of tomato-encoded sRNA as the cross-species antifungal factors targeting the virulence genes of Botrytis cinerea.

Recent evidence shows that small RNAs are transferred from a species to another through cross-species transmission and exhibit biological activities in the receptor. In this study, we focused on tomato-derived sRNAs play a role of defense against Botrytis cinerea. Bioinformatics method was firstly employed to identify tomato-encoded sRNAs as the cross-species antifungal factors targeting B. cinerea genes. Then the expression levels of some identifed sRNAs were checked in B. cinerea-infected plant using qRT-PCR method. Exogenic RNA-induced gene silences analysis were performed to investigate the antifungal roles of the sRNAs, and the target genes in B. cinerea of antifungal sRNAs would be confirmed by using co-expression analysis. Results showed that a total of 21 B.cinerea-induced sRNAs with high abundance were identified as the cross-kingdom regulator candidates. Among them, three sRNAs containing a miRNA (miR396a-5p) and two siRNA (siR3 and siR14) were selected for experimental validation and bioassay analysis. qRT-PCR confirmed that all of these 3 sRNAs were induced in tomato leaves by B. cinerea infection. Correspondingly, 4 virulence genes of B. cinerea respectively targeted by these 3 sRNAs were down-regulated. Bioassay revealed that all of these 3 cross-species sRNAs could inhibit the virulence and spore gemination of B. cinerea. Correspondingly, the coding genes of B. cinerea targeted by these sRNAs were also down-regulated. Moreover, the virulence inhibition by double strand sRNA was more effective than that by single strand sRNA. The inhibition efficiency of sRNA against B. cinerea increased with the increase of its concentration. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived sRNAs to control pathogens.

Salinity variation modulates cellular stress response to ZnO nanoparticles in a sentinel marine bivalve, the blue mussel Mytilussp.

Zinc oxide nanoparticles are released into marine environments from industrial, medical and consumer uses sparking concerns about their potential ecotoxicological effects. Ecological hazard assessment of nZnO in marine ecosystems is hindered by the lack of understanding of the potential interactive effects of nZnO toxicity with other common abiotic stressors, such as salinity fluctuations, in marine organisms. To close this gap in our knowledge, we carried out a comprehensive biomarker-based assessment of the combined effects of salinity and nZnO in a sentinel marine bivalve, the blue mussels Mytilus edulis. The mussels were exposed for 21 days to clean seawater (control), an environmentally relevant concentration (100 µg Zn l-1) of nZnO or dissolved Zn (to identify the toxic effects attributable to Zn2+ toxicity) under the normal (15), low (5) and fluctuating (5-15) salinity regimes. The selected molecular and biochemical markers focused on the oxidative stress, apoptosis, detoxification system and inflammation in the gills and the digestive gland of the mussels. Biomarker analysis showed different effects of nZnO and dissolved Zn on biomarkers of oxidative stress, xenobiotic detoxification and apoptosis but similar effects of both pollutants on the levels of metallothioneins and inflammatory markers. Exposure to nZnO led to elevated levels of lipid peroxidation, upregulation of p53 and p38 stress kinases and apoptosis-related genes, most notably in the gills. Exposure to dissolved Zn led to accumulation of protein carbonyls and activated redox-sensitive detoxification enzymes (NADPH-P450 reductase and glutathione-S-transferase) in the mussels. The ambient salinity had significant effects the cellular adverse effects of nZnO in the mussels. The nZnO-induced cellular stress was detectable under the normal (15) and fluctuating (5-15) salinity conditions in the studied brackish water population of the mussels. At low salinity (5), nZnO toxicity signal was almost completely dampened. These findings indicate that chronic osmotic stress close to the tolerance limits of M. edulis prevails over the effects of the environmentally relevant nZnO and dissolved Zn concentrations in combined exposures. These stressor interactions might ameliorate the cellular toxicity of nZnO in the mussels but limit applicability of cellular stress biomarkers for detecting the toxic effects of nanopollutants in low salinity habitats.

Interactive effects of ZnO nanoparticles and temperature on molecular and cellular stress responses of the blue mussel Mytilus edulis.

Temperature is an important abiotic factor that modulates all aspects of ectotherm physiology, including sensitivity to pollutants. Nanoparticles are emerging pollutants in coastal environments, and their potential to cause toxicity in marine organisms is a cause for concern. Here we studied the interactive effects of temperature (including seasonal and experimental warming) on sublethal toxicity of ZnO nanoparticles (nano-ZnO) in a model marine bivalve, the blue mussel Mytilus edulis. Molecular markers were used to assess the pollutant-induced cellular stress responses in the gills and the digestive gland of mussels exposed for 21 days to 10 µg l-1 and 100 µg l-1 of nano-ZnO or dissolved Zn under different temperature regimes including ambient temperature (10 °C and 15 °C in winter and summer, respectively) or experimental warming (+5 °C). Exposure to high concentration (100 µg l-1) of nano-ZnO caused oxidative injury to proteins and lipids and induced a marked apoptotic response indicated by increased transcript levels of apoptosis-related genes p53, caspase 3 and the MAPK pathway (JNK and p38) and decreased mRNA expression of anti-apoptotic Bcl-2. No significant induction of inflammatory cytokine-related response (TGF-ß and NF-κB) of tissues was observed in nano-ZnO exposed-mussels. Furthermore, the oxidative injury and apoptotic response could differentiate the effects of nano-ZnO from those of dissolved Zn in the mussels. This study revealed that oxidative stress and stress-related transcriptional responses to nano-ZnO were strongly modified by warming and season in the mussels. No single biomarker could be shown to consistently respond to nano-ZnO in all experimental groups, which implies that multiple biomarkers are needed to assess nano-ZnO toxicity to marine organisms under the variable environmental conditions of coastal habitats.

Molecular mechanism of modulating miR482b level in tomato with botrytis cinerea infection.

BACKGROUND: Plant miRNAs are involved in the response to biotic and abiotic stresses by altering their expression levels, and they play an important role in the regulation of plant resistance to stress. However, the molecular mechanism that regulates the expression levels of miRNAs in plants with biotic and abiotic stress still needs to be explored. Previously, we found that the expression of the miR482 family was changed in tomato infected by Botrytis cinerea. In this study, we investigated and uncovered the mechanism underlying the response of miR482 to B. cinerea infection in tomato. RESULTS: First, RT-qPCR was employed to detect the expression patterns of miR482b in tomato infected by B. cinerea, and results showed that miR482b primary transcripts (pri-miR482b) were up-regulated in B. cinerea-infected leaves, but the mature miR482b was down-regulated. Subsequently, we used rapid amplification cDNA end method to amplify the full-length of pri-miR482b. Result showed that the pri-miR482b had two isoforms, with the longer one (consisting 300 bp) having an extra fragment of 53 bp in the 3'-end compared with the shorter one. In vitro Dicer assay indicated that the longer isoform pri-miR482b-x1 had higher efficiency in the post-transcriptional splicing of miRNA than the shorter isoform pri-miR482b-x2. In addition, the transcription level of mature miR482b was much higher in transgenic Arabidopsis overexpressing pri-miR482b-x1 than that in OE pri-miR482b-x2 Arabidopsis. These results confirmed that this extra 53 bp in pri-miR482b-x1 might play a key role in the miR482b biogenesis of post-transcription processing. CONCLUSIONS: Extra 53 bp in pri-miR482b-x1 enhanced miR482b biogenesis, which elevated the transcription level of miR482b. This study clarified the response of miR482 to B. cinerea infection in tomato, thereby helping us further understand the molecular mechanisms that regulate the expression levels of other miRNAs.

Immune responses to ZnO nanoparticles are modulated by season and environmental temperature in the blue mussels Mytilus edulis.

Increased production and release of ZnO nanoparticles (nZnO) can cause toxic effects on marine ecosystems and aquatic organisms. However, nZnO toxicity and its modulation by common environmental stressors such as temperature are not yet fully understood. We examined the responses of immune cells (hemocytes) of the blue mussels (Mytilus edulis) exposed to different concentrations (0, 10, 100 µg l-1) of nZnO or dissolved zinc combined with two temperatures (ambient (10 °C in winter and 15 °C in summer) and warming (+5 °C above ambient temperature)) in winter and summer for 21 days. In winter mussels, exposure to nZnO induced a strong transcriptomic response in multiple immune and inflammation-related genes, stimulated phagocytosis and hemocyte mortality yet suppressed adhesion capacity of hemocytes. In summer mussels, the immune cell responses to nZnO were blunted. The transcriptional responses of hemocytes to dissolved Zn were qualitatively similar but weaker than the responses to nZnO. In the absence of the toxic stress, +5 °C warming lead to dysregulation of the transcription of key immune-related genes in the summer but not the winter mussels. Seasonal warm acclimatization and additional warming in summer suppressed the nZnO-induced transcriptional upregulation of antimicrobial peptides, Toll-like receptors and the complement system. These findings demonstrate that nZnO act as an immunogen in M. edulis and indicate that +5 °C warming might have detrimental effect on innate immunity of the temperate mussel populations in summer when exposure to pathogens is especially high. Capsule: ZnO nanoparticles act as an immunotoxicant inducing a strong immune response in the mussels which is dysregulated by warming in summer but not in winter.

Salinity-dependent effects of ZnO nanoparticles on bioenergetics and intermediate metabolite homeostasis in a euryhaline marine bivalve, Mytilus edulis.

Engineered nanoparticles including ZnO nanoparticles (nZnO) are important emerging pollutants in aquatic ecosystems creating potential risks to coastal ecosystems and associated biota. The toxicity of nanoparticles and its interaction with the important environmental stressors (such as salinity variation) are not well understood in coastal organisms and require further investigation. Here, we examined the interactive effects of 100 µg l-1 nZnO or dissolved Zn (as a positive control for Zn2+ release) and salinity (normal 15, low 5, and fluctuating 5-15) on bioenergetics and intermediate metabolite homeostasis of a keystone marine bivalve, the blue mussel Mytilus edulis from the Baltic Sea. nZnO exposures did not lead to strong disturbances in energy or intermediate metabolite homeostasis regardless of the salinity regime. Dissolved Zn exposures suppressed the mitochondrial ATP synthesis capacity and coupling as well as anaerobic metabolism and modified the free amino acid profiles in the mussels indicating that dissolved Zn is metabolically more damaging than nZnO. The environmental salinity regime strongly affected metabolic homeostasis and altered physiological and biochemical responses to nZnO or dissolved Zn in the mussels. Exposure to low (5) or fluctuating (5-15) salinity affected the physiological condition, energy metabolism and homeostasis, as well as amino acid metabolism in M. edulis. Generally, fluctuating salinity (5-15) appeared bioenergetically less stressful than constantly hypoosmotic stress (salinity 5) in M. edulis indicating that even short (24 h) periods of recovery might be sufficient to restore the metabolic homeostasis in this euryhaline species. Notably, the biological effects of nZnO and dissolved Zn became progressively less detectable as the salinity stress increased. These findings demonstrate that habitat salinity must be considered in the biomarker-based assessment of the toxic effects of nanopollutants on coastal organisms.

Season-dependent effects of ZnO nanoparticles and elevated temperature on bioenergetics of the blue mussel Mytilus edulis.

Input of ZnO nanoparticles (nZnO) from multiple sources have raised concerns about the potential toxic effects on estuarine and coastal organisms. The toxicity of nZnO and its interaction with common abiotic stressors (such as elevated temperature) are not well understood in these organisms. Here, we examined the bioenergetics responses of the blue mussel Mytilus edulis exposed for 21 days to different concentrations of nZnO or dissolved zinc (Zn2+) (0, 10, 100 µg l-1) and two temperatures (ambient and 5 °C warmer) in winter and summer. Exposure to nZnO had little effect on the protein and lipid levels, but led to a significant depletion of carbohydrates and a decrease in the electron transport system (ETS) activity. Qualitatively similar but weaker effects were found for dissolved Zn. In winter mussels, elevated temperature (15 °C) led to elevated protein and lipid levels increasing the total energy content of the tissues. In contrast, elevated temperature (20 °C) resulted in a decrease in the lipid and carbohydrate levels and suppressed ETS in summer mussels. These data indicate that moderate warming in winter (but not in summer) might partially compensate for the bioenergetics stress caused by nZnO toxicity in M. edulis from temperate areas such as the Baltic Sea.

Screening value for gastrointestinal lesions of magnetic-controlled capsule endoscopy in asymptomatic individuals.

BACKGROUND AND AIM: Most patients with gastric tumors and precancerous lesions are asymptomatic, which often results in delayed diagnosis and treatment. Compared with conventional gastroscopy and capsule endoscopy, magnetic-controlled capsule endoscopy is a non-invasive, effective, and cost-efficient diagnostic modality for gastric examination. We retrospectively investigated magnetic-controlled capsule endoscopy as a screening tool for gastrointestinal lesions (particularly gastric tumors and precancerous lesions) in asymptomatic individuals. METHODS: In this retrospective study, 1757 patients who voluntarily underwent magnetic-controlled capsule endoscopy between January and December 2019 at nine medical centers across Shaanxi province based on strict inclusion and exclusion criteria were enrolled. The primary outcomes were gastric tumor and precancerous lesion detection rates and procedural safety. RESULTS: The upper and lower gastrointestinal lesion detection rates were 98.35% (1728/1757) and 21.61% (78/361), respectively; 2.28% of patients were diagnosed with gastric tumors including gastric cancer (4/1757) and submucosal tumors (36/1757). Three types of precancerous lesions were found in 591 patients (33.64%), including chronic atrophic gastritis (23.16%), gastric polyp (10.98%), and gastric ulcer (2.96%). For patients aged over 40 years, the detection rate of precancerous lesions was higher (14.36% vs 42.58%, P < 0.001). No patient was diagnosed with small intestinal cancer. No adverse events occurred. CONCLUSIONS: Magnetic-controlled capsule endoscopy could be used as a promising novel screening modality for diagnosis of gastrointestinal lesions in asymptomatic individuals, specifically gastric tumors and precancerous lesions, with the advantages of safety, non-invasiveness, effectiveness, and cost-efficiency.

miR319c acts as a positive regulator of tomato against Botrytis cinerea infection by targeting TCP29.

miR319 family is one of the oldest and most conservative miRNA families in plant and plays an important role in plant development and abiotic stress response. In our previous study, the abundance of sly-miR319c was increased in tomatoes infected by B. cinerea, but the roles and regulatory mechanisms of sly-miR319c in B. cinerea-infected tomato remain unclear. In this study, we confirmed that miR319c was increased in tomato with B. cinerea infection. In contrast, A TCP transcript factor, TCP29, targeted by sly-miR319c was decreased in B. cinerea-infected tomato. Therefore, transgenic Arabidopsis overexpressing sly-miR319c or its target were generated for understanding the biological roles and molecular mechanism of miR319c in B.cinerea-infected plants. Results showed that miR319c overexpression improved the resistance of transgenic plants to B. cinerea, whereas TCP29 overexpression increased the susceptibility of transgenic plant to B. cinerea. So far, TCP transcription factors have been reported mainly in developmental processes. Our data indicate that TCP29 act as a negative regulator to B.cinerea infection. In conclusion, our results indicate that sly-miR319c is a positive regulator of tomato resistance to B. cinerea infection by targeting TCP29.

Novel tomato miRNA miR1001 initiates cross-species regulation to suppress the conidiospore germination and infection virulence of Botrytis cinerea in vitro.

Recent evidence has shown that microRNAs are transferred from one species to another through cross-species transmission and exhibit biological activities in the receptor. However, the cross-kingdom regulation of pathogen virulence by plant-derived miRNAs is rarely reported. This study investigated the regulatory role of novel tomato miRNA miR1001 in the growth and development of Botrytis cinerea. Results showed that miR1001 inhibited the virulence of B. cinerea-infected plants, and the inhibitory effect of miR1001/miR1001* was stronger than that of miR1001. Moreover, miR1001 exerted a significant inhibitory effect on the conidiospore germination of B. cinerea. Degradome-seq experiment showed that miR1001 can directly target the Bcin03g02170.1 and Bcin10g01400.1 genes, which respectively encode the ATP-dependent metallopeptidase and cysteine-type endopeptidase, in B. cinerea. The interactions of both targets with miR1001 were further confirmed by using transient co-expression in tobacco. Real-time RT-PCR analysis showed that the expression levels of the two target genes were significantly downregulated in B. cinerea with miR1001 treatment. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived miRNAs to control pathogens.

Toxic epidermal necrolysis syndrome induced by tigecycline: a case report.

A 56-year-old man diagnosed with non-Hodgkin's lymphoma underwent autologous bone marrow transplantation. He was subsequently admitted to the hospital with fever, and his symptoms were initially controlled by multiple antibiotics, including tigecycline. He then developed a generalized body rash that improved after treatment with anti-allergy drugs and steroids. Furthermore, tigecycline treatment for a second time resulted in a severe skin reaction with systemic symptoms, suggesting toxic epidermal necrolysis syndrome (TEN). The patient was shown to have the slow-metabolizing cytochrome P450 2C19 allele, denoted CYP2C19*2. He was transferred to a laminar flow ward and given strict mucosal care, together with corticosteroids and intravenous immunoglobulin. He recovered after 3 weeks of treatment. Tigecycline-induced Stevens-Johnson syndrome (SJS)/TEN has rarely been reported in the Chinese population. However, our experience suggests that Asians are more likely to have adverse reactions to drugs metabolized by the cytochrome P450 enzyme. Early identification of drug reactions and immediate cessation of the suspected drug is essential. Additionally, a combined therapy scheme and a clean laminar flow environment may improve the cure rate of SJS/TEN.

Interactive effects of salinity variation and exposure to ZnO nanoparticles on the innate immune system of a sentinel marine bivalve, Mytilus edulis.

ZnO nanoparticles (nZnO) are released into the coastal environment from multiple sources, yet their toxicity to marine organisms is not well understood. We investigated the interactive effects of salinity (normal 15, low 5, and fluctuating 5-15) and nZnO (100 µg l-1) on innate immunity of the blue mussels Mytilus edulis from a brackish area of the Baltic Sea. Exposure to ionic Zn (100 µg l-1) was used to test whether the toxic effects of nZnO can be attributed to the potential release of Zn2+. Functional parameters and the expression of key immune-related genes were investigated in the mussels exposed to nZnO or ionic Zn under different salinity regimes for 21 days. nZnO exposures elevated hemocyte mortality, suppressed adhesion, stimulated phagocytosis, and led to an apparent increase in lysosomal volume. At salinity 15, nZnO suppressed the mRNA expression of the Toll-like receptors TLRb and c, C-lectin, and the complement system component C3q indicating impaired ability for pathogen recognition. In contrast, the mRNA levels of an antimicrobial peptide defensin increased during nZnO exposure at salinity 15. At fluctuating salinity (5-15), nZnO exposure increased expression of multiple immune-related genes in hemocytes including the complement system components C1 and C3q, and the Toll-like receptors TLRa, b and c. Low salinity (5) had strong immunosuppressive effects on the functional and molecular immune traits of M. edulis that overshadowed the effects of nZnO. The salinity-dependent modulation of immune response to nZnO cannot be attributed to the differences in the aggregation or solubility of nZnO, and likely reflects the interaction of the toxic effects of nanoparticles and physiological effects of the osmotic stress. These findings have implications for the environmental risk assessment of nanomaterials and the development of the context-specific biomarker baselines for coastal pollution monitoring.

Specific dynamic action of mussels exposed to TiO2 nanoparticles and seawater acidification.

Both nanoparticles (NPs) and ocean acidification (OA) pose threats to marine animals as well as marine ecosystems. The present study aims to evaluate the combined effects of NPs and OA on specific dynamic action (SDA) of mussels. The thick shell mussels Mytilus coruscus were exposed to two levels of pH (7.3 and 8.1) and three concentrations of TiO2 NPs (0, 2.5, and 10 mg L-1) for 14 days followed by a 7-day recovery period. The SDA parameters, including standard metabolic rate, peak metabolic rate, aerobic metabolic scope, SDA slope, time to peak, SDA duration and SDA, were measured. The results showed that TiO2 NPs and low pH significantly affected all parameters throughout the experiment. When the mussels were exposed to seawater acidification or TiO2 NPs conditions, standard metabolic rate, aerobic metabolic scope, SDA slope and SDA significantly decreased, whereas peak metabolic rate, time to peak and SDA duration significantly increased. In addition, interactive effects between TiO2 NPs and pH were observed in SDA parameters except time to peak and SDA. Therefore, the synergistic effect of TiO2 NPs and low pH can adversely affect the feeding metabolism of mussels.

Nano-TiO2 impairs digestive enzyme activities of marine mussels under ocean acidification.

With the development of nanotechnology and increased nanomaterial application, TiO2 nanoparticles have been released into the aquatic environment, causing potential ecotoxicological effects on aquatic organisms. Ocean acidification caused by anthropogenic CO2 is one of the most common environmental stressors, occurring simultaneously with marine contaminants, e.g., nanoparticles. Marine bivalves can accumulate nanoparticles and their digestive functions may be affected. In this study, we investigated the potential influences of TiO2 nanoparticles on the digestive enzyme activities of marine mussels Mytilus coruscus under ocean acidification. Mussels were exposed to combined treatments with three concentrations of nano-TiO2 (0, 2.5, 10 mg/L) and two pH values (8.1, 7.3) for 14 days, and then recovered under ambient condition (pH 8.1 and no nanoparticle) for 7 days. Samples were taken on the 1st, 3rd, 7th, 14th, and 21st day, the digestive enzymes, including amylase, pepsin, trypsin, lipase, and lysozyme, were investigated. Our results showed that nano-TiO2 and low pH had negative effects on amylase, pepsin, trypsin, and lipase, while both of them led an increase in lysozyme activity. Nano-TiO2 showed greater effects on the digestive capacity of M. coruscus rather than low pH. Moreover, a recovery period of 7 days was not sufficient for these enzymes to fully recover.

The effects of ZnO nanostructures of different morphology on bioenergetics and stress response biomarkers of the blue mussels Mytilus edulis.

Biofouling causes massive economical losses in the maritime sector creating an urgent need for effective and ecologically non-harmful antifouling materials. Zinc oxide (ZnO) nanorod coatings show promise as an antifouling material; however, the toxicity of ZnO nanorods to marine organisms is not known. We compared the toxicity of suspended ZnO nanorods (NR) with that of ZnO nanoparticles (NP) and ionic Zn2+ in a marine bivalve Mytilus edulis exposed for two weeks to 10 or 100 µg Zn L-1 of ZnO NPs, NRs or Zn2+, or to immobilized NRs. The multi-biomarker assessment included bioenergetics markers (tissue energy reserves, activity of mitochondrial electron transport system and autophagic enzymes), expression of apoptotic and inflammatory genes, and general stress biomarkers (oxidative lesions, lysosomal membrane stability and metallothionein expression). Exposure to ZnO NPs, NRs and Zn2+ caused accumulation of oxidative lesions in proteins and lipids, stimulated autophagy, and led to lysosomal membrane destabilization indicating toxicity. However, these responses were not specific for the form of Zn (NPs, NR or Zn2+) and showed no monotonous increase with increasing Zn concentrations in the experimental exposures. No major disturbance of the energy status was found in the mussels exposed to ZnO NPs, NRs, or Zn2+. Exposure to ZnO NPs and NRs led to a strong induction of apoptosis- and inflammation-related genes, which was not seen in Zn2+ exposures. Based on the integrated biomarker response, the overall toxicity as well as the pro-apoptotic and pro-inflammatory action was stronger in ZnO NPs compared with the NRs. Given the stability of ZnO NR coatings and the relatively low toxicity of suspended ZnO NR, ZnO NR coating might be considered a promising low-toxicity material for antifouling paints.

Classification and characterization of hemocytes from two Asian horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda.

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.