A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species.

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.

Microfluidic chip electrophoresis for biochemical analysis.

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics-based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.

Antioxidant response of the hard shelled mussel Mytilus coruscus exposed to reduced pH and oxygen concentration.

Ocean acidification (OA) and hypoxic events are increasing worldwide problems, their interactive effects have not been well clarified, although their co-occurrence is prevalent. The East China Sea (the Yangtze River estuary area) suffers from not only coastal hypoxia but also pH fluctuation, representing an ideal study site to explore the combined effect of OA and hypoxia on marine bivalves. We experimentally evaluated the antioxidant response of the mussel Mytilus coruscus exposed to three pH levels (8.1, 7.7 and 7.3) at two dissolved oxygen (DO) levels (2.0mgL-1 and 6.0mgL-1) for 72h. Activities of superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase and levels of malondialdehyde were measured in gills and hemolymph. All enzymatic activities in hemolymph and gills followed a similar pattern throughout the experiment duration. Generally, low DO showed greater effects on enzyme activities than elevated CO2. Significant interactions between DO, pH and time were only observed at superoxide dismutase and catalase in both tissues. PCA revealed positive relationships between most enzyme activities in both gills and hemolymph with the exception of alkaline phosphatase activity and the level of malondialdehyde in the hemolymph. Overall, our results suggested that decreased pH and low DO induced similar antioxidant responses in the hard shelled mussel, and showed an additive effect on most enzyme activities. The evaluation of multiple environmental stressors, a more realistic scenario than single ones, is crucial to predict the effect of future global changes on coastal species and our results supply some insights on the potential combined effects of reduced pH and DO on marine bivalves.

An overview of current research on cancer stem cells: a bibliometric analysis.

BACKGROUND: Cancer stem cells (CSCs) represent a potential mechanism contributing to tumorigenesis, metastasis, recurrence, and drug resistance. The objective of this study is to investigate the status quo and advancements in CSC research utilizing bibliometric analysis. METHODS: Publications related to CSCs from 2010 to 2022 were collected from the Web of Science Core Collection database. Various analytical tools including CiteSpace, VOSviewer, Scimago Graphica, and GraphPad Prism were used to visualize aspects such as co-authorship, co-occurrence, and co-citation within CSC research to provide an objective depiction of the contemporary status and developmental trajectory of the CSC field. RESULTS: A total of 22,116 publications were included from 1942 journals written by 95,992 authors. Notably, China emerged as the country with the highest number of publications, whereas the United States exerted the most significant influence within the field. MD Anderson Cancer Center emerged as the institution making the most comprehensive contributions. Wicha M.S. emerged as the most prolific and influential researcher. Among journals, Cancers emerged as a focal point for CSC research, consistently publishing a wealth of high-quality papers. Furthermore, it was observed that most journals tended to approach CSC research from molecular, biological, and immunological perspectives. The research into CSCs encompassed a broad array of topics, including isolation and enrichment techniques, biomarkers, biological characteristics, cancer therapy strategies, and underlying biological regulatory mechanisms. Notably, exploration of the tumor microenvironment and extracellular vesicles emerged as burgeoning research frontiers for CSCs. CONCLUSION: The research on CSCs has garnered growing interest. A trend toward multidisciplinary homogeneity is emerging within the realm of CSCs. Further investigation could potentially center on the patients of extracellular vesicles and the tumor microenvironment in relation to CSCs.

High-resolution history of oxygen depletion in the SW Baltic Sea since the mid-19th century as revealed by bivalve shells.

The Baltic Sea serves as a model region to study processes leading to oxygen depletion. Reconstructing past low-oxygen occurrences, specifically hypoxia, is crucial to understand current ecological disturbances and developing future mitigation strategies. The history of dissolved oxygen (DO) concentration in some Baltic Sea basins has been investigated in previous studies, but temporally well-constrained, inter-annual and better resolved DO reconstructions are still scarce. Here, we present precisely dated, high-resolution DO record since the mid-19th century reconstructed from Mn/Cashell values of Arctica islandica (Bivalvia) collected in the Mecklenburg Bight. According to the data, this area experienced similar low oxygenation during the second half of the 19th century and the late 20th century, but DO variability increased: A 12-15-yr oscillation prevailed in the 19th century, but a 4-6-year period dominated in the late 20th century. Shortly after the onset of the Industrial Revolution around 1850, Mn/Cashell values increased, indicating a DO decrease, probably caused by strong anthropogenic nutrient input. More recently, phosphate levels and inflows of oxygen-rich North Sea water have been identified as major factors controlling the bottom water oxygenation. For example, the increase in DO in the mid-1990s was linked to the decrease in phosphate content and several Major Baltic Inflows. The strong Ba/Cashell rise between the 1860s and the turn of the century most likely reflects changes in diatom community structure rather than a bloom of mass phytoplankton. This is supported by largely unchanged Mn/Cashell and shell growth. Decadal and multi-decadal cycles of shell growth rate correlated strongly with the Atlantic Multidecadal Variability, likely reflecting changes in atmospheric circulation patterns, precipitation rate and riverine nutrient supply. To further improve the management and protection of ecosystems in the Baltic Sea, a larger number of such high-resolution retrospective studies covering long periods of time and large regions are needed.

Microplastics can aggravate the impact of ocean acidification on the health of mussels: Insights from physiological performance, immunity and byssus properties.

Ocean acidification may increase the risk of disease outbreaks that would challenge the future persistence of marine organisms if their immune system and capacity to produce vital structures for survival (e.g., byssus threads produced by bivalves) are compromised by acidified seawater. These potential adverse effects may be exacerbated by microplastic pollution, which is forecast to co-occur with ocean acidification in the future. Thus, we evaluated the impact of ocean acidification and microplastics on the health of a mussel species (Mytilus coruscus) by assessing its physiological performance, immunity and byssus properties. We found that ocean acidification and microplastics not only reduced hemocyte concentration and viability due to elevated oxidative stress, but also undermined phagocytic activity of hemocytes due to lowered energy budget of mussels, which was in turn caused by the reduced feeding performance and energy assimilation. Byssus quality (strength and extensibility) and production were also reduced by ocean acidification and microplastics. To increase the chance of survival with these stressors, the mussels prioritized the synthesis of some byssus proteins (Mfp-4 and Mfp-5) to help maintain adhesion to substrata. Nevertheless, our findings suggest that co-occurrence of ocean acidification and microplastic pollution would increase the susceptibility of bivalves to infectious diseases and dislodgement risk, thereby threatening their survival and undermining their ecological contributions to the community.

Rapid and robust whole slide imaging based on LED-array illumination and color-multiplexed single-shot autofocusing.

BACKGROUND: Digital pathology is experiencing an exponential period of growth catalyzed by advancements in imaging hardware and progresses in machine learning. The use of whole slide imaging (WSI) for digital pathology has recently been cleared for primary diagnosis in the US. The demand for using frozen section procedure for rapid identification of cancerous tissue during surgery is another driving force for the development of WSI. A conventional WSI system scans the tissue slide to different positions and acquires the digital images. In a typical implementation, a focus map is created prior to the scanning process, leading to significant overhead time and a necessity for high positional accuracy of the mechanical system. The resulting cost of WSI system is often prohibitive for frozen section procedure during surgery. METHODS: We report a novel WSI scheme based on a programmable LED array for sample illumination. In between two regular brightfield image acquisitions, we acquire one additional image by turning on a red and a green LED for color multiplexed illumination. We then identify the translational shift of the red- and green-channel images by maximizing the image mutual information or cross-correlation. The resulting translational shift is used for dynamic focus correction in the scanning process. Since we track the differential focus during adjacent acquisitions, there is no positional repeatability requirement in our scheme. RESULTS: We demonstrate a prototype WSI platform with a mean focusing error of ~0.3 microns. Different from previous implementations, this prototype platform requires no focus map surveying, no secondary camera or additional optics, and allows for continuous sample motion in the focus tracking process. CONCLUSIONS: A programmable LED array can be used for color-multiplexed single-shot autofocusing in WSI. The reported scheme may enable the development of cost-effective WSI platforms without positional repeatability requirement. It may also provide a turnkey solution for other high-content microscopy applications.

Differential in vivo hemocyte responses to nano titanium dioxide in mussels: Effects of particle size.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in various products and inevitably released with different sizes and forms into aquatic environment. The purpose of this study was to assess the differential immune toxicity of TiO2 NPs with size difference on mussel hemocytes using flow cytometry (FCM) assays. Hemocyte parameters, including total hemocyte count (THC), hemocyte mortality (HM), phagocytosis activity (PA), lysosomal content (LC), esterase activity (EA), mitochondrial number (MN), mitochondrial membrane potential (MMP) and reactive oxygen species content (ROS) were evaluated in the mussels Mytilus coruscus exposed to two types of TiO2 NPs (25nm & 100nm: 0.1, 1, 10 mg/L, respectively). In general, size- and concentration-dependent toxicity was pronounced with 25nm-NP and highest concentration (10mg/L) being the most toxic. Alhough a slight recovery from the TiO2 exposure was observed, significant carry-over effects were still detected. These results highlight the importance of differential size effects of metal oxide NPs on toxicity mechanisms in aquatic animals.

Combined effects of toxic Microcystis aeruginosa and hypoxia on the digestive enzyme activities of the triangle sail mussel Hyriopsis cumingii.

Nowadays, eutrophication is a very popular environmental problem in numerous waters around the world. The main reason of eutrophication is the enrichment of the nutrient, which results in the excessive growth of phytoplankton and some of them are toxic and harmful. Fortunately, some studies have shown that some bivalves can filter the overgrown phytoplankton in water, which may alleviate water eutrophication. However, the physiological effects of toxic cyanobacteria on filter feeding animal have not been clarified very well. In this experiment, digestive enzyme activities in Hyriopsis cumingii exposed to different concentrations of the toxic Microcystis aeruginosa (0, 5 * 105 and 5 *106 cell ml-1) at two dissolved oxygen (DO) levels (6 and 2 mg l-1) for 14 days were investigated. Toxic M. aeruginosa significantly affected all digestive enzyme activities throughout the experiment. At high toxic M. aeruginosa concentration, the activities of cellulase, amylase and lipase in digestive gland and stomach were significantly increased (P<0.05). However, hypoxia reduced the activities of cellulase, amylase and lipase in digestive gland and stomach. Conflicting effects were observed between toxic M. aeruginosa and DO in most digestive enzyme activities during the exposure period. Therefore, it is not conducive for the digestion and absorption of M. aeruginosa in H. cumingii under hypoxic conditions. H. cumingii is tolerant to toxic M. aeruginosa and may remove toxic cyanobacteria from waters under normal DO conditions.

Transgenerational effects of short-term exposure to acidification and hypoxia on early developmental traits of the mussel Mytilus edulis.

Transgenerational effects of multiple stressors on marine organisms are emerging environmental themes. We thus experimentally tested for transgenerational effects of seawater acidification and hypoxia on the early development traits of the mussel Mytilus edulis. Fertilization rate, embryo deformity rate, and larval shell length were negatively impacted by acidification, while hypoxia had little effect except for increasing deformity rates under control pH conditions. Offspring from low pH/O2 parents were less negatively affected by low pH/O2 conditions than offspring from control parents; however, low pH/O2 conditions still negatively affected developmental traits in offspring from acclimated parents compared to control seawater conditions. Our results demonstrate that experimental seawater acidification and hypoxia can adversely affect early developmental traits of M. edulis and that parental exposure can only partially alleviate these impacts. If experimental observations hold true in nature, it is unlikely that parental exposure will confer larval tolerance to ocean acidification for M. edulis.

Nitrogen fertilization modifies organic transformations and coatings on soil biogeochemical interfaces through microbial polysaccharides synthesis.

The soil-water interfaces (SWI) in soil pores are hotspots for organic matter (OM) transformation. However, due to the heterogeneous and opaque nature of soil microenvironment, direct and continuous tracing of interfacial reactions, such as OM transformations and formation of organo-mineral associations, are rare. To investigate these processes, a new soil microarray technology (SoilChips) was developed and used. Homogeneous 800-µm-diameter SoilChips were constructed by depositing a dispersed Oxisol A horizon suspension on a patterned glass. Dissolved organic matter from the original soil was added on the SoilChips to mimic SWI processes. The effects of ammonium fertilization (90 mg N kg-1 soil) on chemical composition of SWIs were evaluated via X-ray photoelectron spectroscopy. Over 21 days, ammonium addition increased OM coatings at SWIs and modified the OM chemical structure with more alcoholic- and carboxylic-C compared to the unfertilized control. Molecular modeling of OM composition at SWIs showed that N fertilization mainly facilitated the microbial production of glucans. We demonstrated that N availability modifies the specific OM molecular processing and its immobilization on SWIs, thereby providing a direct insight into biogeochemical transformation of OM at micro-scale.

Seawater acidification and temperature modulate anti-predator defenses in two co-existing Mytilus species.

The effects of short-term (7 days) experimental ocean acidification (-0.4 pH units) and warming (+5 °C) on anti-predator defenses of two sympatric Mytilus species from China, M. coruscus and M. edulis, in the presence and absence of predator cues were investigated. Results suggested species-specific independent negative effects of acidification and warming on the number and weight of byssal threads, the force of thread attachment, and total thread plaque area. Similar negative effects were observed for clustering behaviour, with acidification and warming independently increasing the number of solitary individuals and decreasing the percentage of mussels in clusters. Acidification effects on byssus were strongly exacerbated when predators were present. Ultimately, this study suggests that short-term exposure to experimental warming and acidification can negatively impact anti-predator defense strategies in mussels with potential ramifications for predator-prey interactions and ecological functioning in systems where mussel beds play a key ecological role.

Effects of copper on hemocyte parameters in the estuarine oyster Crassostrea rivularis under low pH conditions.

With the development of industry and agriculture, the metal pollutants (e.g., Cu) are inevitably released into the aquatic environment. In addition, ocean acidification (OA) as a major environmental stress is affecting marine organisms. In this study, we investigated the hemocyte responses of the estuarine oyster Crassostrea rivularis exposed to six combinations of two pH levels (8.1 and 7.7) and three Cu concentrations (0, 10 and 50 µg/l) using flow cytometry in vitro and in vivo. In both experiments, Cu and low pH jointly affected the hemocyte parameters of oyster. High Cu exposure resulted in decreased total hemocyte count (THC), esterase activity (EA) and lysosomal content (LC) and increased hemocyte mortality (HM), phagocytosis activity (PA) and reactive oxygen species (ROS) production, especially under low pH conditions. The immune suppression of metal-exposure was more significant than low pH exposure with a 28-d experimental period in oysters. A slight recovery of the immune parameters was observed in THC, HM, PA, ROS and LC. During the depuration period, the modulatory effects of pH were still obvious. In addition, carry-over effects of high Cu and low pH were still observed. Overall, our results showed that copper and low pH weaken immune functions of hemocyte in oysters, with synergistic effects. This work provides new evidence of sublethal negative effects of metals on marine animals under global change scenarios, and copper likely leads to reduced fitness of oysters under low pH conditions.

Synergistic Effects of Nano-ZnO and Low pH of Sea Water on the Physiological Energetics of the Thick Shell Mussel Mytilus coruscus.

In order to investigate the ecotoxicological effects of nano-ZnO particles and seawater acidification on marine bivalves, the thick shell mussels, Mytilus coruscus were subjected to joint treatments with different nano-ZnO concentrations (0 [control], 2.5 [medium] and 10 mg L-1 [high]) under two pH levels (7.7 [low]and 8.1 [control]) for 14 days. The results showed that respiration rate (RR), absorption efficiency (AE), clearance rate (CR), O:N ratio and scope for growth (SFG) were significantly reduced with nano-ZnO concentration increase, but ammonium excretion rate (ER) was increased. Low pH significantly reduced CR, RR, SFG, and O:N ratio of the mussels especially under high nano-ZnO conditions, and significantly increased ER. Principal component analysis (PCA) showed consistent relationships among most tested parameters, especially among SFG, RR, O:N ratio and CR under the normal pH and 0 nano-ZnO conditions. Therefore, seawater acidification and nano-ZnO interactively impact the ecophysiological responses of mussels and cause more severe effects when they appear concurrently.

Salinity mediates the toxic effect of nano-TiO2 on the juvenile olive flounder Paralichthys olivaceus.

Increased production of engineered nanoparticles has raised extensive concern about the potential toxic effects on marine organisms living in estuarine and coastal environments. Meanwhile, salinity is one of the key environmental factors that may influence the physiological activities in flatfish species inhabiting in those waters due to fluctuations caused by freshwater input or rainfall. In this study, we investigated the oxidative stress and histopathological alteration of the juvenile Paralichthys olivaceus exposed to nano-TiO2 (1 and 10 mg L-1) under salinities of 10 and 30 psu for 4 days. In the gills, Na+-K+-ATPase activity significantly deceased after 4 days 10 psu exposure without nano-TiO2 compared with 1 day of acclimating the salinity from the normal salinity (30 psu) to 10 psu. Under this coastal salinity, low concentration (1 mg L-1) of nano-TiO2 exerted significant impacts. In the liver, the activities of superoxide dismutase, catalase, the levels of lipid peroxide and malondialdehyde increased with nano-TiO2 exposed under 30 psu. Such increase indicated an oxidative stress response. The result of the integrated biomarker responses showed that P. olivaceus can be adversely affected by high salinity and high concentration of nano-TiO2 for a short-term (4 days) exposure. The histological analysis revealed the accompanying severe damages for the gill filaments. Principal component analysis further showed that the oxidative stress was associated with the nano-TiO2 effect at normal salinity. These findings indicated that nano-TiO2 and normal salinity exert synergistic effects on juvenile P. olivaceus, and low salinity plays a protective role in its physiological state upon short-term exposure to nano-TiO2. The mechanism of salinity mediating the toxic effects of NPs on estuarine fish should be further considered.

Oxidative stress induced by titanium dioxide nanoparticles increases under seawater acidification in the thick shell mussel Mytilus coruscus.

Biochemical responses of the mussel Mytilus coruscus exposed to different concentrations of titanium dioxide nanoparticles (nano-TiO2) (0, 2.5, 10 mg L-1) and two pH levels (pH 8.1 and pH 7.3) for 14 days. Mussel responses were also investigated after a 7 days recovery period (pH 8.1 and no nanoparticle). Exposure to nano-TiO2 led changes in antioxidant indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH)), biotransformation enzyme activity (GST) and malondialdehyde level (MDA) in gills and digestive glands. An increase in MDA level and a decrease in SOD and GSH activities were observed in gill of mussels exposed to 10 mg L-1 nano-TiO2. This effect was more severe in mussels kept at pH 7.3 as compared to pH 8.1. A different response was observed in the digestive gland as SOD, CAT and GSH levels increased in mussels exposed to nano-TiO2. These contrasting results in digestive glands and gills were only evident at high concentration of nano-TiO2 and low pH. A 7 days recovery period was not sufficient to fully restore SOD, GPx, GST, GSH and MDA levels to levels before exposure to nano-TiO2 and low pH. Overall, our results confirmed that seawater acidification modulates effects of nanoparticles in mussels, and that gills are more sensitive to these stressors as compared with digestive glands.

Short-Term Exposure of Mytilus coruscus to Decreased pH and Salinity Change Impacts Immune Parameters of Their Haemocytes.

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.

Combined effects of ZnO NPs and seawater acidification on the haemocyte parameters of thick shell mussel Mytilus coruscus.

Flow cytometry was used to investigate the immune parameters of haemocytes in thick-shell mussel Mytilus coruscus exposed to different concentrations of ZnO nanoparticles (NPs) (0, 2.5, and 10mgl-1) at two pH levels (7.3 and 8.1) for 14days following a recovery period of 7days. ZnO NPs significantly affected all of the immune parameters throughout the experiment. At high ZnO NPs concentrations, total haemocyte counting, phagocytosis, esterase, and lysosomal content were significantly decreased whereas haemocyte mortality and reactive oxygen species (ROS) were increased. Although low pH also significantly influenced all of the immune parameters of the mussels, its effect was not as strong as that of ZnO NPs. Interactive effects were observed between pH and ZnO NPs in most haemocyte parameters during the exposure period. Although a slight recovery from the stress of ZnO NPs and pH was observed for all immune parameters, significant carry-over effects of low pH and ZnO NPs were still detected. This study revealed that high concentration of ZnO NPs and low pH exert negative and synergistic effects on mussels, and these effects remain even after the mussels are no longer exposed to such stressors.

Impact of zinc oxide nanoparticles and ocean acidification on antioxidant responses of Mytilus coruscus.

Increased production of engineered nanoparticles (NPs) has raised extensive concerns about the potential toxic effects on marine organisms. Extensive evidences documented the impact of ocean acidification (OA) on the physiology and fitness of bivalves. In the present study, we investigated the biochemical responses of the mussel Mytilus coruscus exposed to both nano-ZnO and low pH relevant for ocean acidification conditions for 14 d followed by a 7-d recovery period. Most biochemical indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), acid phosphatase (ACP) and alkaline phosphatase (ALP)) measured in gills and hemocytes were increased when the mussels were subject to low pH or high concentration of nano-ZnO, suggesting oxidative stress responses. No significant interactions between the two stressors were observed for most measured parameters. After a 1 week recovery period, low pH and nano-ZnO had less marked impact for SOD, GPx, ACP and ALP in hemocytes as compared to the end of the 14 d exposure. However, no recovery was observed in gills. Overall, our results suggest that both low pH and nano-ZnO induce an anti-oxidative response in Mytilus coruscus with gills being more sensitive than hemocytes.

CO2-induced pH reduction increases physiological toxicity of nano-TiO2 in the mussel Mytilus coruscus.

The increasing usage of nanoparticles has caused their considerable release into the aquatic environment. Meanwhile, anthropogenic CO2 emissions have caused a reduction of seawater pH. However, their combined effects on marine species have not been experimentally evaluated. This study estimated the physiological toxicity of nano-TiO2 in the mussel Mytilus coruscus under high pCO2 (2500-2600 µatm). We found that respiration rate (RR), food absorption efficiency (AE), clearance rate (CR), scope for growth (SFG) and O:N ratio were significantly reduced by nano-TiO2, whereas faecal organic weight rate and ammonia excretion rate (ER) were increased under nano-TiO2 conditions. High pCO2 exerted lower effects on CR, RR, ER and O:N ratio than nano-TiO2. Despite this, significant interactions of CO2-induced pH change and nano-TiO2 were found in RR, ER and O:N ratio. PCA showed close relationships among most test parameters, i.e., RR, CR, AE, SFG and O:N ratio. The normal physiological responses were strongly correlated to a positive SFG with normal pH and no/low nano-TiO2 conditions. Our results indicate that physiological functions of M. coruscus are more severely impaired by the combination of nano-TiO2 and high pCO2.