The hidden risk of microplastic-associated pathogens in aquatic environments.

Increasing studies of plastisphere have raised public concern about microplastics (MPs) as vectors for pathogens, especially in aquatic environments. However, the extent to which pathogens affect human health through MPs remains unclear, as controversies persist regarding the distinct pathogen colonization on MPs as well as the transmission routes and infection probability of MP-associated pathogens from water to humans. In this review, we critically discuss whether and how pathogens approach humans via MPs, shedding light on the potential health risks involved. Drawing on cutting-edge multidisciplinary research, we show that some MPs may facilitate the growth and long-range transmission of specific pathogens in aquatic environments, ultimately increasing the risk of infection in humans. We identify MP- and pathogen-rich settings, such as wastewater treatment plants, aquaculture farms, and swimming pools, as possible sites for human exposure to MP-associated pathogens. This review emphasizes the need for further research and targeted interventions to better understand and mitigate the potential health risks associated with MP-mediated pathogen transmission.

Toxicological assessment of cadmium-containing quantum dots in developing zebrafish: Physiological performance and neurobehavioral responses.

The present research investigated the effects of exposure to sublethal concentrations of cadmium selenide/zinc sulfide (CdSecore/ZnSshell)-containing quantum dots (QDs; 0 - 100 µg/L QDs) on the neurophysiological performance of developing zebrafish (Danio rerio). The results suggested that exposure to CdSe QDs for 5 days increased the whole-body content of Cd without affecting the general physiological conditions of larvae. Interestingly, CdSe QD exposure reduced swimming distance but increased swimming velocity of larvae, suggesting that the exposure may lead to burst/episodic swimming. The findings also suggested that CdSe QD exposure reduced the wall-hugging behavior of larvae during a sudden light-to-dark transition test, and that the exposure significantly decreased the locomotor activity of fish during the dark period. On the other hand, control larvae displayed a dark avoidance behavior, whereas CdSe QD-exposed larvae exhibited an increase in the time spent in the dark zone, providing further support that CdSe QDs inhibited anxiety-related responses in larvae. Additional analysis with droplet digital PCR revealed that CdSe QD exposure altered the mRNA levels of genes that are associated with dopamine signaling and oxidative stress response. Collectively, our findings suggested that CdSe QD exposure may induce neurobehavioural toxicity and alters the mRNA abundance of dopamine- and oxidative stress-related genes in developing animals.

Early developmental exposure to bisphenol A and bisphenol S disrupts socio-cognitive function, isotocin equilibrium, and excitation-inhibition balance in developing zebrafish.

Dysregulation of the oxytocinergic system and excitation/inhibition (E/I) balance in synaptic transmission and neural circuits are common hallmarks of various neurodevelopmental disorders. Several experimental and epidemiological studies have shown that perinatal exposure to endocrine-disrupting chemicals bisphenol A (BPA) and bisphenol S (BPS) may contribute to a range of childhood neurodevelopmental disorders. However, the effects of BPA and BPS on social-cognitive development and the associated mechanisms remain largely unknown. In this study, we explored the impacts of early developmental exposure (2hpf-5dpf) to environmentally relevant concentrations of BPA, and its analog BPS (0.001, 0.01, and 0.1 µM), on anxiety, social behaviors, and memory performance in 21 dpf zebrafish larvae. Our results revealed that early-life exposure to low concentrations of BPA and BPS elevated anxiety-like behavior, while fish exposed to higher concentrations of these chemicals displayed social deficits and impaired object recognition memory. Additionally, we found that co-exposure with an aromatase inhibitor antagonized BPA- and BPS-induced effects on anxiety levels and social behaviors, while the co-exposure to an estrogen receptor antagonist restored recognition memory in zebrafish larvae. These results indicate that BPA and BPS may affect social-cognitive function through distinct mechanisms. On the other hand, exposure to low BPA/BPS concentrations increased both the mRNA and protein levels of isotocin (zebrafish oxytocin) in the zebrafish brain, whereas a reduction in its mRNA level was observed at higher concentrations. Further, alterations in the transcript abundance of chloride transporters, and molecular markers of gamma-aminobutyric acid (GABA) and glutamatergic systems, were observed in the zebrafish brain, suggesting possible E/I imbalance following BPA or BPS exposure. Collectively, the results of this study demonstrate that early-life exposure to low concentrations of the environmental contaminants BPA and BPS can interfere with the isotocinergic signaling pathway and disrupts the establishment of E/I balance in the developing brain, subsequently leading to the onset of a suite of behavioral deficits and neurodevelopmental disorders.

Functional significance and physiological regulation of essential trace metals in fish.

Trace metals such as iron, copper, zinc and manganese play essential roles in various biological processes in fish, including development, energy metabolism and immune response. At embryonic stages, fish obtain essential metals primarily from the yolk, whereas in later life stages (i.e. juvenile and adult), the gastrointestine and the gill are the major sites for the acquisition of trace metals. On a molecular level, the absorption of metals is thought to occur at least in part via specific metal ion transporters, including the divalent metal transporter-1 (DMT1), copper transporter-1 (CTR1), and Zrt- and Irt-like proteins (ZIP). A variety of other proteins are also involved in maintaining cellular and systemic metal homeostasis. Interestingly, the expression and function of these metal transport- and metabolism-related proteins can be influenced by a range of trace metals and major ions. Increasing evidence also demonstrates an interplay between the gastrointestine and the gill for the regulation of trace metal absorption. Therefore, there is a complex network of regulatory and compensatory mechanisms involved in maintaining trace metal balance. Yet, an array of factors is known to influence metal metabolism in fish, such as hormonal status and environmental changes. In this Review, we summarize the physiological significance of iron, copper, zinc and manganese, and discuss the current state of knowledge on the mechanisms underlying transepithelial metal ion transport, metal-metal interactions, and cellular and systemic handling of these metals in fish. Finally, we identify knowledge gaps in the regulation of metal homeostasis and discuss potential future research directions.

Algal Organic Matter Drives Methanogen-Mediated Methylmercury Production in Water from Eutrophic Shallow Lakes.

Algal blooms bring massive amounts of algal organic matter (AOM) into eutrophic lakes, which influences microbial methylmercury (MeHg) production. However, because of the complexity of AOM and its dynamic changes during algal decomposition, the relationship between AOM and microbial Hg methylators remains poorly understood, which hinders predicting MeHg production and its bioaccumulation in eutrophic shallow lakes. To address that, we explored the impacts of AOM on microbial Hg methylators and MeHg production by characterizing dissolved organic matter with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy and quantifying the microbial Hg methylation gene hgcA. We first reveal that the predominance of methanogens, facilitated by eutrophication-induced carbon input, could drive MeHg production in lake water. Specifically, bioavailable components of AOM (i.e., CHONs such as aromatic proteins and soluble microbial byproduct-like materials) increased the abundances (Archaea-hgcA gene: 438-2240% higher) and activities (net CH4 production: 16.0-44.4% higher) of Archaea (e.g., methanogens). These in turn led to enhanced dissolved MeHg levels (24.3-15,918% higher) for three major eutrophic shallow lakes in China. Nevertheless, our model results indicate that AOM-facilitated MeHg production could be offset by AOM-induced MeHg biodilution under eutrophication. Our study would help reduce uncertainties in predicting MeHg production, providing a basis for mitigating the MeHg risk in eutrophic lakes.

Influence of Microplastics on the Mobility, Bioavailability, and Toxicity of Heavy Metals: A Review.

Microplastics (MPs) can pose ecological risk to the environment and have the potential to negatively affect human health, raising serious public concerns. It is recognized that MPs could act as a vector for various environmental pollutants including heavy metals and potentially influencing their mobility, fate, and bioavailabilty in the environment. However, knowledge on the mechanisms underpinning the interaction processes between MPs and heavy metals is far from clear. This review discusses the effects of MPs on the adsorption/desorption, speciation and bioavailability, and toxicity of various heavy metals. The present review also systematically identifies the environmental factors (e.g., pH, ionic strength, and organic matters) that could affect their interaction processes. This work aims to establish a meaningful perspective for a comprehensive understanding of the indirect ecological risks of MPs as vectors for contaminants. The work also provides a reference for the development of better regulatory strategies in mitigating the negative effects caused by the co-existence of MPs and heavy metals.

Using zebrafish as a model to assess the individual and combined effects of sub-lethal waterborne and dietary zinc exposure during development.

The present research used zebrafish (5-28 days post-fertilization; dpf) as a model organism to investigate the effects of chronic exposure to environmentally relevant sub-lethal concentrations of waterborne (261 µg/L) and dietary zinc (Zn) (1500 mg Zn/kg dw), either independently or simultaneously, during development. The results showed that whole body contents of Zn were increased in all Zn treatment groups, with the highest accumulation of Zn observed in larvae simultaneously exposed to elevated waterborne and dietary Zn. In addition, exposure to elevated levels of Zn, either through the water or the diet, led to a decrease in whole body calcium (Ca) contents at 28 dpf. The findings also suggested that exposure to elevated levels of Zn resulted in a significant reduction in whole body manganese (Mn) contents. More importantly, the magnitude of decrease in Mn contents by Zn exposure was markedly higher than that in Ca and appeared to mirror the increases in whole body Zn accumulation. These results indicate that Mn regulation is more sensitive than Ca to disruption by Zn exposure in developing fish. Further examination of the Zrt-Irt-Like Protein (ZIP) family of transporters using droplet digital PCR technologies revealed that several zip transporters exhibited temporal and exposure route-specific changes following Zn exposure. In particular, the level of zip4 was influenced by Zn exposure regardless of the exposure routes, while changes in zip7 and zip8 levels were predominantly driven by waterborne exposure. Overall, our findings demonstrated that zebrafish during the developmental periods are sensitive to elevated levels of Zn seen in the environment, particularly following co-exposures to waterborne and dietary Zn. Future toxicological assessment of elevated Zn exposure should consider both the exposure routes and the life stages of fish.

How do humans recognize and face challenges of microplastic pollution in marine environments? A bibliometric analysis.

Microplastics (MPs) are abundant in marine environments, drawing global attention from scientists and rendering it significant to review the research progress and predict future trends of this field. To achieve that, we collected 1898 publications on marine MPs from Web of Science and performed a bibliometric analysis by CiteSpace and VOSviewer. Additionally, we utilized an unrestricted retrieval of literature from ScienceDirect to supplement our major findings. Trends in publication numbers show the growth in study from the initial stage ( 2012 and before), when microplastic (MP) occurrence, abundance, and distribution were primarily investigated. Throughout the ascent stage (between 2013-2016), when diverse sampling and analytical methods were applied to capture and identify MPs from the ocean, baseline data have been gleaned on physiochemical properties of MPs. The research focus then shifted to the bioaccumulation and ecotoxicological effects of MPs on marine biota, further highlighting their potential deleterious impacts on human health via dietary exposure, and this period was defined as the exploration stage (2017 and onwards). Nevertheless, key challenges including the lack of standard procedures for MP sampling, technical limitations in MP detecting and identification, and controversy about the underlying effects on the marine ecosystems and humans have also been arisen in the last decade. The present study elucidates how we gradually recognize MP pollution in marine environments and what challenges we face, suggesting future avenues for MP research.

Understanding the effects of sulfur input on mercury methylation in rice paddy soils.

Sulfur could be introduced into paddy soils via dry or wet deposition, irrigation, and fertilization, which subsequently impacts the production of methylmercury (MeHg), a bioaccumulative neurotoxicant. However, effects of sulfur input on MeHg production are variable, possibly due to the multiple effects of sulfur on Hg mobility and/or microbial Hg methylators, leading to uncertainties in predicting MeHg risk. To address that, we explored the effects of different types and amounts of sulfur as well as concentrations of ambient sulfate on Hg methylation in paddy soils, and elucidated the mechanisms by quantifying changes in (1) Hg mobility and (2) microbial Hg methylators (e.g., sulfate-reducing bacteria, SRB). Our results indicated that MeHg levels increased by 40-86% and 30-96% in soils under various types (i.e., 200 mg kg-1 elemental sulfur, ammonium sulfate, sulfur-coated urea and potassium sulfate (K2SO4)) and different amounts (i.e., 100, 200 and 400 mg kg-1 K2SO4) of sulfur input. The enhanced MeHg production could be explained by increased Hg mobility but not changes in microbial Hg methylators. Besides, sulfate input increased MeHg levels (89-240%) in soils with low ambient sulfate levels (<100 mg kg-1) but had no effect on high-sulfate soils (>380 mg kg-1). These could be explained by the diverse responses of Hg mobility and microbial Hg methylators to sulfate input at different ambient sulfate levels. Our study opens the "black box" of Hg methylation under sulfur input, which would help reduce uncertainties in predicting MeHg risk in soils.

A comprehensive review on the neuropathophysiology of selenium.

As an essential micronutrient, selenium (Se) exerts its biological function as a catalytic entity in a variety of enzymes. From a toxicological perspective, however, Se can become extremely toxic at concentrations slightly above its nutritional levels. Over the last few decades, there has been a growing level of concern worldwide regarding the adverse effects of both inorganic and organic Se compounds on a broad spectrum of neurological functions. A wealth of evidence has shown that exposure to excess Se may compromise the normal functioning of various key proteins, neurotransmitter systems (the glutamatergic, dopaminergic, serotonergic, and cholinergic systems), and signaling molecules involved in the control and regulation of cognitive, behavioral, and neuroendocrine functions. Elevated Se exposure has also been suspected to be a risk factor for the development of several neurodegenerative and neuropsychiatric diseases. Nonetheless, despite the various deleterious effects of excess Se on the central nervous system (CNS), Se neurotoxicity and negative behavioral outcomes are still disregarded at the expense of its beneficial health effects. This review focuses on the current state of knowledge regarding the neurobehavioral effects of Se and discusses its potential mode of action on different aspects of the central and peripheral nervous systems. This review also provides a brief history of Se discovery and uses, its physicochemical properties, biological roles in the CNS, environmental occurrence, and toxicity. We also review potential links between exposure to different forms of Se compounds and aberrant neurobehavioral functions in humans and animals, and identify key knowledge gaps and hypotheses for future research.

Straw return enhances the risks of metals in soil?

Interactions between organic matter (OM) and metals in soils are important natural mechanisms that can mitigate metal bioaccumulation in terrestrial environments. A primary source of OM in soils is straw return, accounting for more than 65% of OM input. Straw-OM has long been believed to reduce metal bioaccumulation, e.g., by immobilizing metals in soils. However, there is growing evidence that straw return could possibly enhance bioavailability and thus risks (i.e., food safety) of some metals in crops, including Cd, Hg, and As. Poor understanding of straw return-induced increases in metal bioavailability would add uncertainty in assessing or mitigating risks of metals in contaminated farming soils. Here, 863 pieces of literature (2000-2019) that reported the effects of straw return on metal bioavailability and bioaccumulation were reviewed. Mechanisms responsible for the increased metal mobility and bioavailability under straw return are summarized, including the effects of dissolution, complexation, and methylation. Effects of straw return on the physiology and the absorption of metals in plants is also discussed (i.e., physiological effect). These mechanisms are then used to explain the observed increases in the mobility, bioavailability, and bioaccumulation of Cd, Hg, and As under straw amendment. Information summarized in this study highlights the importance to re-consider the current straw return policy, particularly in metal-contaminated farmlands.

Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio).

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish (Danio rerio) is expressed abundantly in Na+-absorbing/H+-secreting H+-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a-/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a-/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na+ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na+ content remained constant. While Cl- uptake was significantly reduced in ca17a-/- mutants, Cl- content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl- uptake, implicating Ca17a in the mechanism of Cl- uptake by larval zebrafish. H+ secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a-/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

The neurophysiological effects of iron in early life stages of zebrafish.

Trace metal/ion homeostasis, neurophysiological performance, and molecular responses to iron (Fe) exposure were investigated in the model organism zebrafish (Danio rerio). The findings demonstrated that exposure to a sublethal concentration of ferric iron (Fe3+) increased Fe contents in both the whole body and head region of developing zebrafish. Among the various trace metals and major ion examined, a dysregulation in manganese, zinc, nickel, and calcium balance was also observed in Fe-exposed larvae. Further biochemical assay and in-vivo imaging revealed that Fe exposure resulted in possible oxidative stress-induced damage, and an increased generation of reactive oxygen species in specific regions of the larvae. Using a droplet digital PCR (ddPCR) technology, it was found that the expression levels of various oxidative stress-responsive genes were temporally modulated by Fe exposure. Additionally, Fe-exposed larvae exhibited an impairment in escape response and a decrease in swimming activity. These larvae also appeared to exhibit a reduced anxiety-like behaviour. Together, our research suggested that larvae experiencing an increased Fe loading exhibited a dysregulation in metal homeostasis and a decrease in neurophysiological performance. These results suggested that neurophysiological assessments are sensitive methods to evaluate Fe toxicity in developing fish.

A comprehensive review of the neurobehavioral effects of bisphenol S and the mechanisms of action: New insights from in vitro and in vivo models.

The normal brain development and function are delicately driven by an ever-changing milieu of steroid hormones arising from fetal, placental, and maternal origins. This reliance on the neuroendocrine system sets the stage for the exquisite sensitivity of the central nervous system to the adverse effects of endocrine-disrupting chemicals (EDCs). Bisphenol A (BPA) is one of the most common EDCs which has been a particular focus of environmental concern for decades due to its widespread nature and formidable threat to human and animal health. The heightened regulatory actions and the scientific and public concern over the adverse health effects of BPA have led to its replacement with a suite of structurally similar but less known alternative chemicals. Bisphenol S (BPS) is the main substitute for BPA that is increasingly being used in a wide array of consumer and industrial products. Although it was considered to be a safe BPA alternative, mounting evidence points to the deleterious effects of BPS on a wide range of neuroendocrine functions in animals. In addition to its reproductive toxicity, recent experimental efforts indicate that BPS has a considerable potential to induce neurotoxicity and behavioral dysfunction. This review analyzes the current state of knowledge regarding the neurobehavioral effects of BPS and discusses its potential mode of actions on several aspects of the neuroendocrine system. We summarize the role of certain hormones and their signaling pathways in the regulation of brain and behavior and discuss how BPS induces neurotoxicity through interactions with these pathways. Finally, we review potential links between BPS exposure and aberrant neurobehavioral functions in animals and identify key knowledge gaps and hypotheses for future research.

Regulation of metal homeostasis and zinc transporters in early-life stage zebrafish following sublethal waterborne zinc exposure.

In the present research, the effects of exposure to a sublethal concentration of zinc (Zn) on metal and ion homeostasis, and the regulation and the localization of various Zn transporters (i.e., the Zrt-Irt Like Protein (ZIP) family of Zn transporters), were investigated in zebrafish (Danio rerio) during early development. Exposure to an elevated level of Zn [4 µM (high) vs. 0.25 µM (control)] from 0 day post-fertilization (dpf) resulted in a significant increase in the whole body content of Zn at 5 dpf. A transient decrease in the whole body calcium (Ca) level was observed in 3 dpf larvae exposed to high Zn. Similarly, whole body nickel (Ni) and copper (Cu) contents were also reduced in 3 dpf larvae exposed to high Zn. Importantly, the magnitude of reduction in whole body Ni and Cu contents following Zn exposure was markedly higher than that in Ca content, suggesting that internal Ni and Cu balance were likely more sensitive to Zn exposure in developing zebrafish. Exposure to high Zn altered the mRNA expression levels of specific zip transporters, with an increase in zip1 (at 3 dpf) and zip8 (at 5 dpf), and a decrease in zip4 (at 5 dpf). The expression levels of most zip transporters tended to decrease from 3 dpf to 5 dpf with the exception of zip4 and zip8. Results from in situ hybridization revealed that several zip transporters exhibited distinct spatial distribution (e.g., zip8 in the intestinal tract, zip14 in the pronephric tubules). Overall, our findings suggested that exposure to sublethal concentrations of Zn disrupts the homeostasis of essential metals during early development and that different ZIP transporters may play unique roles in regulating Zn homeostasis in various organs in developing zebrafish.

Influence of dietary iron exposure on trace metal homeostasis and expression of metal transporters during development in zebrafish☆.

The present study investigated the effects of dietary iron (Fe) exposure on physiological performance and homeostatic regulation of trace metals during development (5-28 days post-fertilization; dpf) in zebrafish (Danio rerio). The results demonstrated that whole body Fe content was increased in 14 dpf larvae fed a high Fe diet. Cumulative mortality was also significantly elevated during exposure to the high Fe diet. Using droplet digital PCR, we observed that high Fe-exposed larvae exhibited an increase in mRNA levels of the Fe-storage protein ferritin, which appeared to be associated with the elevated level of whole body Fe content. Further, the results indicated that dietary Fe exposure induced transient changes in the mRNA expression levels of various metal transporters, including the iron transporter dmt1, and the zinc transporters zip8 and zip14. The expression of the epithelial Ca2+ channels (i.e., ecac) was also found to increase by high dietary Fe. Overall, our findings suggest that larval fish during the early nutritional transition period are sensitive to the effects of dietary Fe.

Cadmium exposure reduces the density of a specific ionocyte subtype in developing zebrafish.

The present study examined the effects of waterborne cadmium (Cd) exposure on ionic balance and ionocyte density in developing zebrafish (Danio rerio) (0-4 days post-fertilization). Fish exposed to 1 or 10 µg Cd/L exhibited an increase in whole body Cd level. Exposure to 10 µg Cd/L also significantly reduced whole body content of Ca2+, but not other major ions (e.g., Na+, K+ and Mg2+). Such reduction was accompanied by a decrease in the density of Ca2+-transporting ionocytes, the Na+/K+-ATPase-rich cells (NaRCs). However, the densities of other ionocyte subtypes (e.g., Na+-transporting ionocytes) remained unchanged after exposure to 10 µg Cd/L. The potential interactive effects between water chemistry and Cd exposure on ionocyte density were examined further in Cd-exposed larvae acclimated to different water NaCl or Ca2+ levels. The results demonstrated that NaRC density increased in fish acclimated to low Ca2+ water, presumably increasing Ca2+ uptake for maintaining Ca2+ homeostasis. However, Cd exposure completely abolished the increased NaRC density in low water Ca2+ environments. The increased NaRCs over development was also reduced in Cd-exposed larvae. In conclusion, our study suggested that Cd exposure reduces the density of NaRCs and suppresses the compensatory regulation of NaRCs during acclimation to low water Ca2+ level. These inhibitory effects by Cd exposure ultimately disrupt Ca2+ balance in the early life stages of zebrafish.

Reassessing the contribution of the Na+/H+ exchanger Nhe3b to Na+ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing.

Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+ exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl--cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol l-1) and 'low' (10 µmol l-1) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+ uptake was sustained in nhe3b KO larvae under both Na+ conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+ uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+ flux by HR cells, did not reduce Na+ uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl--cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+ uptake under normal or low Na+ These findings highlight the flexibility of the Na+ uptake system and demonstrate that Nhe3b is expendable to Na+ uptake in zebrafish and that our understanding of Na+ uptake mechanisms in this species is incomplete.