Reducing Gut Dissolution of Zinc Oxide Nanoparticles by Secondary Microplastics with Consequent Impacts on Barnacle Larvae.

The environmental impact of sunscreen is a growing concern, yet the combined effects of its components on marine animals are poorly understood. In this study, we investigated the combined effects of sunscreen-extracted zinc oxide nanoparticles (nZnO) and microplastics (MPs) on the development of barnacle larvae, focusing on the different roles played by primary microplastics (PMPs) and secondary microplastics (SMPs) generated through the phototransformation of PMPs. Our findings revealed that a lower concentration of nZnO (50 µg/L) enhanced molting and eye development in barnacle larvae, while a higher concentration (500 µg/L) inhibited larval growth. Co-exposure to PMPs had no significant effect on larval development, whereas SMPs mitigated the impact of nZnO by restricting the in vivo transformation to ionic Zn. Accumulated SMPs reduced gut dissolution of nZnO by up to 40%, lowering gut acidity by 85% and buffering the in vivo dissolution of nZnO. We further identified a rough-surfaced Si-5 fragment in SMPs that damaged larval guts, resulting in decreased acidity. Another Si-32 resisted phototransformation and had no discernible effects. Our study presented compelling evidence of the impacts of SMPs on the bioeffect of nZnO, highlighting the complex interactions between sunscreen components and their combined effects on marine organisms.

Single-Cell RNA Sequencing Profiling Cellular Heterogeneity and Specific Responses of Fish Gills to Microplastics and Nanoplastics.

Fish gills are highly sensitive organs for microplastic (MP) and nanoplastic (NP) invasions, but the cellular heterogeneity of fish gills to MPs and NPs remains largely unknown. We employed single-cell RNA sequencing to investigate the responses of individual cell populations in tilapia Oreochromis niloticus gills to MP and NP exposure at an environmentally relevant concentration. Based on the detected differentially expressed gene (DEG) numbers, the most affected immune cells by MP exposure were macrophages, while the stimulus of NPs primarily targeted T cells. In response to MPs and NPs, H+-ATPase-rich cells exhibited distinct changes as compared with Na+/K+-ATPase-rich cells and pavement cells. Fibroblasts were identified as a potential sensitive cell-type biomarker for MP interaction with O. niloticus gills, as evidenced by the largely reduced cell counts and the mostly detected DEGs among the 12 identified cell populations. The most MP-sensitive fibroblast subpopulation in O. niloticus gills was lipofibroblasts. Cell-cell communications between fibroblasts and H+-ATPase-rich cells, neurons, macrophages, neuroepithelial cells, and Na+/K+-ATPase-rich cells in O. niloticus gills were significantly inhibited by MP exposure. Collectively, our study demonstrated the cellular heterogeneity of O. niloticus gills to MPs and NPs and provided sensitive markers for their toxicological mechanisms at single-cell resolution.

Dynamic Regulation of Intracellular Labile Cu(I)/Cu(II) Cycle in Microalgae Chlamydomonas reinhardtii: Disrupting the Balance by Cu Stress.

The labile metal pool involved in intracellular trafficking and homeostasis is the portion susceptible to environmental stress. Herein, we visualized the different intracellular distributions of labile Cu(I) and Cu(II) pools in the alga Chlamydomonas reinhardtii. We first demonstrated that labile Cu(I) predominantly accumulated in the granules within the cytoplasmic matrix, whereas the labile Cu(II) pool primarily localized in the pyrenoid and chloroplast. The cell cycle played an integral role in balancing the labile Cu(I)/Cu(II) pools. Specifically, the labile Cu(II) pool primarily accumulated during the SM phase following cell division, while the labile Cu(I) pool dynamically changed during the G phase as cell size increased. Notably, the labile Cu(II) pool in algae at the SM stage exhibited heightened sensitivity to environmental Cu stress. Exogenous Cu stress disrupted the intracellular labile Cu(I)/Cu(II) cycle and balance, causing a shift toward the labile Cu(II) pool. Our proteomic analysis further identified a putative cupric reductase, potentially capable of reducing Cu(II) to Cu(I), and four putative multicopper oxidases, potentially capable of oxidizing Cu(I) to Cu(II), which may be involved in the conversion between the labile Cu(I) pool and labile Cu(II) pool. Our study elucidated a dynamic cycle of the intracellular labile Cu(I)/Cu(II) pools, which were accessible and responsive to environmental changes.

Novel Near-Infrared-II In Vivo Visualization Revealed Rapid Calcium Intestine Turnover in Daphnia magna with Delayed Impact by Cadmium and Acidification.

Calcium is a highly demanded metal, and its transport across the intestine of Daphnia magna remains a significant unresolved question. Due to technical constraints, the visualization of the kinetic process of Ca passage through D. magna has been challenging. Here, we developed the second near-infrared Ca sensor (NIR-II Ca) and conducted real-time in vivo imaging of Ca in daphnids with a high signal-to-noise ratio, deep tissue penetration, and minimal damage. Through the utilization of the NIR-II Ca sensor, we for the first time visualized and quantified the kinetic process of Ca passage in the intestine in real time. The results revealed that trophically available Ca passed through the intestines in 24 h, whereas waterborne Ca required only 35 min. This rapid "flushing through" mechanism established waterborne Ca as the primary source of Ca absorption. However, environmental stressors such as water acidification and cadmium significantly delayed the Ca passage and absorption. The development of NIR imaging and sensors allows for real-time dynamic visualization of contaminants/nutrients in organisms and holds great potential as a powerful tool for future studies into material kinetic processes in living animals.

Metagenomics-Based Microbial Ecological Community Threshold and Indicators of Anthropogenic Disturbances in Estuarine Sediments.

Assessing the impacts of cumulative anthropogenic disturbances on estuarine ecosystem health is challenging. Using spatially distributed sediments from the Pearl River Estuary (PRE) in southern China, which are significantly influenced by anthropogenic activities, we demonstrated that metagenomics-based surveillance of benthic microbial communities is a robust approach to assess anthropogenic impacts on estuarine benthic ecosystems. Correlational and threshold analyses between microbial compositions and environmental conditions indicated that anthropogenic disturbances in the PRE sediments drove the taxonomic and functional variations in the benthic microbial communities. An ecological community threshold of anthropogenic disturbances was identified, which delineated the PRE sediments into two groups (H and L) with distinct taxa and functional traits. Group H, located nearshore and subjected to a higher level of anthropogenic disturbances, was enriched with pollutant degraders, putative human pathogens, fecal pollution indicators, and functional traits related to stress tolerance. In contrast, Group L, located offshore and subjected to a lower level of anthropogenic disturbances, was enriched with halotolerant and oligotrophic taxa and functional traits related to growth and resource acquisition. The machine learning random forest model identified a number of taxonomic and functional indicators that could differentiate PRE sediments between Groups H and L. The identified ecological community threshold and microbial indicators highlight the utility of metagenomics-based microbial surveillance in assessing the adverse impacts of anthropogenic disturbances in estuarine sediments, which can assist environmental management to better protect ecosystem health.

Physiological and immune profiling of tilapia Oreochromis niloticus gills by high-throughput single-cell transcriptome sequencing.

The physiological and immune functions of fish gills are largely recognized, but their following functional heterogeneity at the single cell scale has been rarely reported. Here, we performed single cell RNA sequencing (scRNA-seq) on the gills of tilapia fish Oreochromis niloticus. We identified a total of 12 cell populations and analyzed their functional heterogeneity. To investigate the physiological function of O. niloticus gills, expression patterns of genes encoding ion transporters were selected from the identified H+-ATPase-rich cells (HR cells), Na+/K+-ATPase-rich cells (NaR cells), and pavement cells. Specific enrichment of ca4a, slc9a1a, and LOC100692482 in the HR cells of O. niloticus gills explained their functions in acid-base regulation. Genes encoding Ca2+ transporters, including atp2b1, LOC100696627, and LOC 100706765, were specifically expressed in the NaR cells. Pavement cells were presumably the main sites responsible for ammonia and urea transports in O. niloticus gills with specific enrichment of Rhbg and LOC100693008, respectively. The expression patterns of the four immune cell subtypes varied greatly, with B cells being enriched with the most immune-related GO terms. KEGG enrichment analysis showed that MAPK signaling pathway was the most enriched pathway among the four types of immune cells in O. niloticus gills. Our results are important in understanding the physiological and immune responses of fish gills at the cellular resolution.

Internalization of the Metal-Organic Framework MIL-101(Cr)-NH2 by a Freshwater Alga and Transfer to Zooplankton.

The common metal-organic framework (MOF) MIL-101(Cr)-NH2 has attracted considerable attention due to its great potential applications in the environmental field. Nevertheless, its behavior and fate in aquatic systems are unknown. This study quantified and visualized the interactions of MIL-101(Cr)-NH2 with the freshwater phytoplanktonic alga Chlamydomonas reinhardtii and its potential trophic transfer to zooplankton. The unicellular alga absorbed and accumulated the MOF by surface attachment, forming agglomerates and eventually cosettling out from water. Bioimaging revealed that MIL-101(Cr)-NH2 was internalized by the algal cells and mainly occurred in the pyrenoid. Without algae in a freshwater system, MIL-101(Cr)-NH2 was ingested by Daphnia magna, showing steadily increasing concentrations approaching 1-9% of dry body weight. Addition of algae substantially suppressed D. magna uptake of MIL-101(Cr)-NH2 by 63.8-97.9%. Such inhibition could be explained by the competitive uptake of MOF by the algae and the inductive effects of algal food on MOF elimination by D. magna. The MOF (≤1 mg/L) ingested by D. magna was centered in the gut regions, whereas large MOF or algae-MOF aggregates were adsorbed onto the carapace and appendages, including the antennae, at 10 mg/L. Overall, the algae were the major targets for MIL-101(Cr)-NH2, with nearly all algal cells settling out at 10 mg/L within 24 h. The possibility of trophic transfer of MIL-101(Cr)-NH2 to D. magna in aquatic systems with algae present was limited due to its low accumulation potential and short retention time in D. magna.

Cu(I)/Cu(II) Released by Cu Nanoparticles Revealed Differential Cellular Toxicity Related to Mitochondrial Dysfunction.

Cu-based nanoparticles (NPs) have been increasingly fabricated, and different Cu species (i.e., Cu+ and Cu2+) of these NPs are tuned to achieve differential physicochemical properties. Although ion release is one of the major toxic mechanisms of Cu-based NPs, differences in cytotoxicity between released Cu(I) and Cu(II) ions are largely unknown. In this study, the A549 cells exhibited a lower tolerance to Cu(I) compared with Cu(II) accumulation. Bioimaging of labile Cu(I) indicated that the change of the Cu(I) level upon CuO and Cu2O exposure displayed different trends. We then developed a novel method to selectively release Cu(I) and Cu(II) ions within the cells by designing CuxS shells for Cu2O and CuO NPs, respectively. This method confirmed that Cu(I) and Cu(II) exhibited different cytotoxicity mechanisms. Specifically, excess Cu(I) induced cell death through mitochondrial fragmentation, which further led to apoptosis, whereas Cu(II) resulted in cell cycle arrest at the S phase and induced reactive oxygen species generation. Cu(II) also led to mitochondrial fusion, which was likely due to the influence of the cell cycle. Our study first uncovered the difference between the cytotoxicity mechanisms of Cu(I) and Cu(II), which could be greatly beneficial for the green fabrication of engineered Cu-based NPs.

Photodegradation of Microplastics by ZnO Nanoparticles with Resulting Cellular and Subcellular Responses.

Both zinc oxide nanoparticles (ZnO NPs) and microplastics (MPs) were extracted from one commercial sunscreen, while other ingredients were removed based on the "like dissolves like" principle. MPs were further extracted by acidic digestion of ZnO NPs using HCl and characterized as spherical particles of approximately 5 µm with layered sheets in an irregular shape on the surface. Although MPs were stable in the presence of simulated sunlight and water after 12 h of exposure, ZnO NPs promoted the photooxidation by producing hydroxyl radicals, with a 2.5-fold increase in the carbonyl index of the degree of surface oxidation. As a result of surface oxidation, spherical MPs were more soluble in water and fragmented to irregular shapes with sharp edges. We then compared the cytotoxicity of primary MPs and secondary MPs (25-200 mg/L) to the HaCaT cell line based on viability loss and subcellular damages. The cellular uptake of MPs transformed by ZnO NPs was enhanced by over 20%, and MPs caused higher cytotoxicity compared with the pristine ones, as evidenced by a 46% lower cell viability, 220% higher lysosomal accumulation, 69% higher cellular reactive oxygen species, 27% more mitochondrial loss, and 72% higher mitochondrial superoxide at 200 mg/L. Our study for the first time explored the activation of MPs by ZnO NPs derived from commercial products and revealed the high cytotoxicity caused by secondary MPs, providing new evidence on the effects of secondary MPs on human health.

Accumulation Kinetics and Gut Microenvironment Responses to Environmentally Relevant Doses of Micro/Nanoplastics by Zooplankton Daphnia Magna.

Microplastics are emerging pollutants that have been widely reported in aquatic ecosystems. Based on the analysis of environmentally relevant concentrations of microplastics in global freshwater systems, herein, we employed aggregated-induced emission (AIE) microplastic fluorogens and imaged and quantified the bioaccumulation of differentially charged micro- (20 µm)/nano- (200 nm) plastics (MNPs) in zooplankton Daphnia magna. We found that all particles of different sizes and charges were readily ingested, especially larger-sized and positively charged MNPs, with over 50% of the ingested particles accumulating in the gut. Bioaccumulation of MNPs reached 50% of steady-state condition within 1 h. The presence of algae inhibited the ingestion and depuration of MNPs. To further demonstrate the effects of such accumulation on gut health, we further applied the AIE probes for visualizing the pH and esterase in the digestive tract, as well as the gut inflammation. An accumulation of MNPs in D. magna significantly and rapidly induced the acidification of gut pH while inducing esterase activity. The NPs apparently induced gut inflammation in contrast to the MPs, demonstrating the size-dependent effects on oxidative stress. Our results highlighted that MNP exposure at environmentally relevant concentrations perturbed the microenvironments of zooplankton guts, which may significantly affect their digestion and assimilation of food materials as well as contaminant uptake.

Lysosomal Cu(I)/Cu(II) Dependence of Antimicrobial Ability of Oyster Hemocytes and Regulation of Phagolysosomal System.

Copper (Cu) is hyperaccumulated in oyster hemocytes and is an essential trace metal indispensable for diverse innate immune functions. However, the roles of Cu in oyster immune defense are still unclear. In this study, Cu exposure enhanced the phagocytosis of zymosan by increasing the number and length of filopodia, as well as mitochondrial ROS (mitoROS) production mainly in granulocytes, followed by semigranulocytes and agranulocytes. The intracellular calcium level increased to promote the phagosome-lysosome fusion after Cu exposure. The enhancement of phagosomal acidification and mitochondrion-phagosome juxtaposition were also found in granulocytes after Cu exposure. These results indicated that Cu could regulate the phagolysosomal system to enhance the antimicrobial ability of oyster hemocytes with the assistance of mitoROS. Furthermore, Cu(I) and Cu(II) were predominately located in lysosomes, and degranulation may provide a mechanism for exposing Cu to bacteria to prevent their survival and proliferation. Specifically, we showed that the newly formed Cu(I) arising from lysosomal Cu(II) moved to lysosomes and mitochondria in activated hemocytes to induce strong immune responses. The ability of the transformation of Cu(I) from Cu(II) followed granulocytes > semigranlocytes > agranulocytes, indicating that granulocytes played important roles in immune functions of oysters. Our results provided new insights into the understanding of antimicrobial effects of Cu in oyster hemocytes.

Tracking Nano- and Microplastics Accumulation and Egestion in a Marine Copepod by Novel Fluorescent AIEgens: Kinetic Modeling of the Rhythm Behavior.

Nano- and microplastics (NMPs) are now prevalent in the marine environment. This study quantified the uptake and depuration kinetics of spherical polystyrene NMPs of different particle sizes (200 nm/30 µm) and functional groups (-NH2/-COOH) in a temperate calanoid copepod Calanus sinicus (C. sinicus), which exhibited rhythmic feeding patterns in natural environments. Aggregated-induced emission (AIE) fluorescent probes were employed to track and quantify the kinetics of NMPs with excellent photostability and biocompatibility. The results showed that C. sinicus consumed all NMPs types, with preference of NMPs to small size and amino group. Increased diatom concentrations also inhibited the bioaccumulation of NMPs. Influenced by rhythmic behavior, the bioaccumulation of NMPs by C. sinicus was nonstationary during the 6 h uptake phase. After 1-3 h of rapid uptake, the body burden peaked and then slowly declined. During the 3 h depuration phase, C. sinicus rapidly and efficiently removed NMPs with a mean half-life of only 0.23 h. To further quantify the body burden of C. sinicus under the influence of rhythmic feeding behavior, a biokinetic model was established, and the Markov chain Monte Carlo method was used to estimate the parameter distribution. Our results highlighted that copepods exhibited unique rhythmic feeding behavior under environmentally relevant concentrations of NMPs exposure, which may influence the bioaccumulation, trophic transfer, and environmental fate of NMPs.

In Situ Tracking of Crystal-Surface-Dependent Cu2O Nanoparticle Dissolution in an Aqueous Environment.

Metal-oxide-based nanoparticles (MONPs) such as Cu2O NPs have attracted growing attention, but the potential discharges of MONPs have raised considerable concern of their environmental fate including their dissolution behavior. The impacts of morphology on MONP dissolution are largely uncertain due to the lack of in situ tracking techniques. In this study, we combined a series of in situ technologies including liquid-cell transmission electron microscopy and fluorescence probes to reveal the in situ dissolution process of Cu2O NPs in freshwater. Our results suggest that cubic Cu2O NPs exhibit a higher dissolution quantity compared with spherical NPs of the same surface area. The difference was mainly related to the crystal surface, while other factors such as particle size or aggregation status showed minor effects. Importantly, we demonstrated the simultaneous growth of new small NPs and the dissolution of pristine Cu2O NPs during the dissolution of Cu2O NPs. Cubic Cu2O NPs became much less soluble under O2-limited conditions, suggesting that O2 concentration largely affected the dependence of dissolution on the NP morphology. Our findings highlight the potential application of in situ techniques to track the environmental fates of MONPs, which would provide important information for assessing the ecological risks of engineered NPs.

Two-Compartmental Toxicokinetic Model Predicts Interspecies Sensitivity Variation of Imidacloprid to Aquatic Invertebrates.

Interspecies sensitivity to the same chemical can be several orders of magnitude different. Quantifying toxicologically internal levels and toxicokinetic (TK) parameters is critical in elucidating the interspecies sensitivity. Herein, a two-compartmental TK model was constructed to characterize the uptake, distribution, and elimination kinetics toward interspecies sensitivity to an insecticide, imidacloprid. Imidacloprid exhibited the highest lethality to the insect Chironomus dilutus, followed by Lumbriculus variegatus, Hyalella azteca, and Daphnia magna. Interspecies sensitivity of imidacloprid to these invertebrates varied by ∼1000 folds based on water concentrations (LC50). Remarkably, the sensitivity variation decreased to ∼50 folds based on the internal residues (LR50), highlighting the critical role of TK in interspecies sensitivity. A one-compartmental TK model failed to simulate the bioaccumulation of imidacloprid in these invertebrates except for D. magna. Instead, a two-compartmental model successfully simulated the slow elimination of imidacloprid in the remaining three species by internally distinguishing the highly dynamic (C1) and toxicologically available (C2) fractions. We further showed that the species sensitivity of the invertebrates to imidacloprid was significantly related to C2, demonstrating that C2 was toxicologically available and responsible for the toxicity of imidacloprid. This mechanistic-based model bridged the internal distribution of organic contaminants in small invertebrates and the associated toxic potency.

Identification of Transformation Products of Organic UV Filters by Photooxidation and Their Differential Estrogenicity Assessment.

Organic ultraviolet filters (OUVFs) are extensively released into aquatic environments, where they undergo complex phototransformation. However, there is little knowledge regarding their transformation products (TPs) and associated endocrine disruption potentials. In the present study, we characterized the chemical and toxicological profiles of TPs for two common OUVFs, oxybenzone (BP3) and ethylhexyl methoxycinnamate (EHMC), by photooxidation under environmentally relevant conditions. It is hypothesized that TPs of the tested OUVFs will show varied estrogenicity at different reaction times. High-resolution liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) identified 17 TPs of 7 m/z for BP-3 and 13 TPs of 8 m/z for EHMC at confidence levels ≤2. Five novel TPs of 2 m/z were reported for the first time with structure-diagnostic MS/MS spectra. Estrogenicity assessment using the MCF-7-luc cell line showed discrepant estrogenic activities exhibited by OUVF-TPs over time. Specifically, BP3-TPs exhibited significantly greater estrogenicity than the parent at several reaction times, whereas EHMC-TPs displayed fluctuating estrogenicity with a declining trend. Correlation analysis coupled with molecular docking simulations further suggested several TPs of BP3 as potential endocrine disruptive compounds. These findings underscore the necessity of considering mixtures during chemical testing and risk assessment and highlight the potentially greater risks associated with post-transformation cocktails.

Functional heterogeneity of immune defenses in molluscan oysters Crassostrea hongkongensis revealed by high-throughput single-cell transcriptome.

Oyster is the worldwide aquaculture molluscan and evolves a complex immune defense system, with hemocytes as the major immune system for its host defense. However, the functional heterogeneity of hemocyte has not been characterized, which markedly hinders our understanding of its defense role. Here, we used the single-cell transcriptome profiling (scRNA-seq), which provides a high-resolution visual insight into its dynamics, to map the hemocyte and assess its heterogeneity in a molluscan oyster Crassostrea hongkongensis. By combining with the cell type specific RNA-seq, thirteen subpopulations belonging to granulocyte, semi-granulocyte, and hyalinocyte were revealed. The granulocytes mainly participated in immune response and autophagy process. Pseudo-temporal ordering of granulocytes identified two different cell-lineages. The hematopoietic transcription factors regulated networks controlling their differentiations were also identified. We further identified one subpopulation of granulocytes in immune activate states with the cell cycle and immune responsive genes expressions, which illustrated the functional heterogeneity of the same cell type. Collectively, our scRNA-seq analysis demonstrated the hemocytes diversity of molluscans. The results are important in our understanding of the immune defense evolution and functional differentiation of hemocytes in Phylum Mollusca.

The inclusion of extract from aerial part of Scutellaria baicalensis in feeding of pearl gentian grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceo-latus♂) promotes growth and immunity.

The root of Scutellaria baicalensis (Scutellaria Radix) has been used as herbal medicine for years in China; however, its stem and leaf (aerial part) are considered as waste. The water extract of aerial part of S. baicalensis, named as SBA, having anti-microbial property has been applied in fish aquaculture. To extend the usage of SBA in fish feeding, SBA was employed to feed pearl gentian grouper (a hybrid of Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂), and subsequently the total fish output, the levels of digestive enzymes and inflammatory cytokines were determined. Feeding the fish with different doses of SBA for two months, the body length and weight were significantly increased by 5%-10%. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver and muscle of SBA-fed fish were doubled, which could account the growth promoting effect of SBA. Besides, the activity of digestive enzyme, lipase, and the expressions of anti-inflammatory cytokines were markedly stimulated by 2-3 times under the feeding of 3% SBA-containing diet. The results indicated the growth promoting activity of SBA in culture of pearl gentian grouper, as well as the effect of SBA in strengthening the immunity. These beneficial effects of SBA feeding can increase the total yield of pearl gentian grouper in aquaculture. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.

Meeting Zn Needs during Medaka Eye Development: Nanoscale Visualization of Retina by Expansion Microscopy.

Fish eyes require high Zn levels to support their early development. Although numerous studies have been conducted on the nutritional and toxic effects of Zn on the eye, the Zn requirement for retinal cell development is still debatable. Moreover, due to the complexity of the retinal structure, it is difficult to clearly visualize each retinal layer and accurately separate cell morphology in vivo by conventional methods. In the present study, we for the first time have achieved nanoscale imaging of retinal anatomy affected by dietary and waterborne Zn exposure by novel expansion microscopy. We demonstrated that the fish retina showed different developmental strategies in response to dietary and aqueous Zn exposures. Excess dietary Zn produced toxicity to retinal photoreceptor cells, resulting in a reduction in cell number and cell area, and this toxicity became severe with biological development. In contrast, waterborne Zn in the natural environment probably failed to meet the Zn requirements of retinal development. Overall, our results indicated that during early development, the Zn requirement of the fish eyes was sensitive, and oversupplementation led to impaired photoreceptor cell development. Our study has provided new perspectives using the powerful and novel expansion microscopy technique in toxicity assessment, enabling ultra-clear visualization of small but complex organ development.

Highly Sensitive and Specific Responses of Oyster Hemocytes to Copper Exposure: Single-Cell Transcriptomic Analysis of Different Cell Populations.

Oyster hemocytes are the primary vehicles transporting and detoxifying metals and are regarded as important cells for the occurrence of colored oysters due to copper (Cu) contamination. However, its heterogeneous responses under Cu exposure have not been studied. Single-cell transcriptome profiling (scRNA-seq) provides high-resolution visual insights into tissue dynamics and environmental responses. Here, we used scRNA-seq to study the responses of different cell populations of hemocytes under Cu exposure in an estuarine oyster Crassostrea hongkongensis. The 1900 population-specific Cu-responsive genes were identified in 12 clusters of hemocytes, which provided a more sensitive technique for examining Cu exposure. The granulocyte, semigranulocyte, and hyalinocyte had specific responses, while the granulocyte was the most important responsive cell type and displayed heterogeneity responses of its two subtypes. In one subtype, Cu was transported with metal transporters and chelated with Cu chaperons in the cytoplasm. Excess Cu disturbed oxidative phosphorylation and induced reactive oxygen species production. However, in the other subtype, endocytosis was mainly responsible for Cu internalization, which was sequestered in membrane-bound granules. Collectively, our results provided the first mRNA expression profile of hemocytes in oysters and revealed the heterogeneity responses under Cu exposure.

Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization.

CuO nanoparticles (NPs) show promising applications in biosensors, waste treatment, and energy materials, but the growing manufacture of CuO NPs also leads to the concerns for their potential environmental and health risks. However, the cellular fates of CuO NPs such as Cu ion dissolution, transformation, and efflux remain largely speculative. In the present study, we for the first time combined the gold-core labeling and Cu ion bioimaging technologies to reveal the intracellular fates of CuO NPs in different cells following cellular internalization of NPs. We demonstrated that the dissolution rate of CuO NPs depended on the cell type. Following CuO dissolution, limited transformation of Cu(II) to Cu(I) occurred within the cellular microenvironment. Instead, Cu(II) was rapidly eliminated from the cells, and such rapid efflux in different cells was highly dependent on the GSH-mediated pathway and lysosome exocytosis. The labile Cu(I) level in the two cancerous cell lines was immediately regulated upon Cu exposure, which explained their tolerance to Au@CuO NPs. Overall, our study demonstrated a very rapid turnover of Cu in the cells following CuO internalization, which subsequently determined the cellular toxicity of CuO. The results will have important implications for assessing the health risk of CuO NPs.