Gulf toadfish (Opsanus beta) urinary bladder ion and water transport is enhanced by acclimation to higher salinity to serve water balance.

Marine teleosts experience ion gain and water loss in their natural habitats. Among other tissues, the urinary bladder epithelium of marine fishes has been shown to actively transport ions to facilitate water absorption. However, transport properties of the urinary bladder epithelium of marine fishes and its plasticity in altered ambient salinities is relatively under-investigated. We describe urinary bladder epithelium electrophysiology, water flux, and expressions of ion transporters in urinary bladder tissue of Gulf toadfish (Opsanus beta) acclimated to either 35 ppt or 60 ppt seawater. Water absorption in bladder sac preparations increased ~ 350% upon acclimation to 60 ppt. Increases in water transport coincided with a significant ~ 137% increase in urinary bladder tissue mucosal-to-serosal short circuit current (Isc) and a ~ 56% decrease in tissue membrane resistance. Collectively, these metrics indicate that an active electrogenic system facilitates water absorption via Na+ (and Cl-) transport in urinary bladder tissue. Furthermore, pharmacological inhibition of urinary bladder tissue Isc and expression of a suite of ion transporters and channels previously unidentified in this tissue provide mechanistic insights into the transport processes responsible for water flux. Analysis of water transport to overall Gulf toadfish water balance reveals a modest water conservation role for the urinary bladder of ~ 0.5% of total water absorption in 35 ppt and 1.9% in 60 ppt acclimated toadfish. These results emphasize that electrogenic ion transport facilitates water-absorptive properties of the urinary bladder in Gulf toadfish - a process that is regulated to facilitate water homeostasis.

Quantification of cytosolic 'free' calcium in isolated coral cells with confocal microscopy.

Despite its prominent role as an intracellular messenger in all organisms, cytosolic free calcium ([Ca2+]i) has never been quantified in corals or cnidarians in general. Ratiometric calcium dyes and cell imaging have been key methods in successful research on [Ca2+]i in model systems, and could be applied to corals. Here, we developed a procedure to quantify [Ca2+]i in isolated cells from the model coral species Stylophora pistillata using Indo-1 and confocal microscopy. We quantified [Ca2+]i in coral cells with and without intracellular dinoflagellate symbionts, and verified our procedure on cultured mammalian cells. We then used our procedure to measure changes in [Ca2+]i in coral cells exposed to a classic inhibitor of [Ca2+]i regulation, thapsigargin, and also used it to record elevations in [Ca2+]i in coral cells undergoing apoptosis. Our procedure paves the way for future studies into intracellular calcium in corals and other cnidarians.

Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic diseases worldwide. Streptococcus mutans, the chief causative agent of caries, uses a 25-kDa manganese-dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression, and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we focused our attention on 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of RNA sequencing revealed 19 sRNAs in S. mutans, which were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 10 sRNAs that were differentially expressed in UA159 cells grown in the presence of low vs high manganese. We describe SmsR1532 and SmsR1785 as SloR- and manganese-responsive sRNAs that are processed from large transcripts and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25-kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterized sRNAs that are both SloR and manganese responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen and that can enable the development of an effective anti-caries therapeutic.

An Ion-Engineering Strategy to Design Hollow FeCo/CoFe2 O4 Microspheres for High-Performance Microwave Absorption.

Although assembled hollow architectures have received considerable attention as lightweight functional materials, their uncontrollable self-aggregation and tedious synthetic methods hinder precise construction and modulation. Therefore, this study proposes a bi-ion synergistic regulation strategy to design assembled hollow-shaped cobalt spinel oxide microspheres. Dominated by the coordination-etching effects of F- and the hydrolysis-complex contributions of NH4 + , the unique construction is formed attributed to the dynamic cycles between metal complexes and precipitates. Meanwhile, their basic structures are perfectly retained after reduction treatment, enabling FeCo/CoFe2 O4 bimagnetic system to be obtained. Subsequently, in-depth analyses are conducted. Investigations reveal that multiscale magnetic coupling networks and enriched air-material heterointerfaces contribute to the remarkable magnetic-dielectric behavior, supported by the advanced off-axis electron holography technique. Consequently, the obtained FeCo/CoFe2 O4 composites exhibit excellent microwave absorption performances with minimal reflection losses (RLmin ) as high as -51.6 dB, an effective absorption bandwidth (EAB) of 4.7 GHz, and a matched thickness of 1.4 mm. Thus, this work provides an informative guide for rationally assembling building blocks into hollow architectures as advanced microwave absorbers through bi-ion and even multi-ion synergistic engineering mechanisms.

In Situ Constructing Robust and Highly Conductive Solid Electrolyte with Tailored Interfacial Chemistry for Durable Li Metal Batteries.

Employing nanofiber framework for in situ polymerized solid-state lithium metal batteries (SSLMBs) is impeded by the insufficient Li+ transport properties and severe dendritic Li growth. Both critical issues originate from the shortage of Li+ conduction highways and nonuniform Li+ flux, as randomly-scattered nanofiber backbone is highly prone to slippage during battery assembly. Herein, a robust fabric of Li0.33 La0.56 Ce0.06 Ti0.94 O3-δ /polyacrylonitrile framework (p-LLCTO/PAN) with inbuilt Li+ transport channels and high interfacial Li+ flux is reported to manipulate the critical current density of SSLMBs. Upon the merits of defective LLCTO fillers, TFSI- confinement and linear alignment of Li+ conduction pathways are realized inside 1D p-LLCTO/PAN tunnels, enabling remarkable ionic conductivity of 1.21 mS cm-1 (26 °C) and tLi+ of 0.93 for in situ polymerized polyvinylene carbonate (PVC) electrolyte. Specifically, molecular reinforcement protocol on PAN framework further rearranges the Li+ highway distribution on Li metal and alters Li dendrite nucleation pattern, boosting a homogeneous Li deposition behavior with favorable SEI interface chemistry. Accordingly, excellent capacity retention of 76.7% over 1000 cycles at 2 C for Li||LiFePO4 battery and 76.2% over 500 cycles at 1 C for Li||LiNi0.5 Co0.2 Mn0.3 O2 battery are delivered by p-LLCTO/PAN/PVC electrolyte, presenting feasible route in overcoming the bottleneck of dendrite penetration in in situ polymerized SSLMBs.

Functional divergence of teleost carbonic anhydrase 4.

The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.

Freshwater adaptation in prickly sculpin (Pisces: Cottidae): intraspecific comparisons reveal evidence for water pH and Na+ concentration driving diversity in gill H+-ATPase and ion regulation.

Phenotypic divergence is a hallmark of adaptive radiation. One example involves differentiation in physiological traits involved in ion regulation among species with contrasting lifestyles and living in distinct environments. Differentiation in ion regulation and its ecological implications among populations within species are, however, less well understood. To address this knowledge gap, we collected prickly sculpin (Cottus asper) from distinct habitat types including coastal rivers connected to estuaries, coastal lakes and interior lakes, all from British Columbia, Canada. We tested for differences in plasma Na+ and Cl-, gill Na+/K+-ATPase and H+-ATPase activity and protein abundance as well as changes in body mass and arterial blood pH in fish sampled from the field and acclimated to two different freshwater conditions in the laboratory: artificial lake water (ALW) and ion-poor water (IPW). We also tested for links between environmental water chemistry and the physiological characteristics associated with ion regulation. Transfer to IPW resulted in upregulation of gill Na+/K+-ATPase and H+-ATPase activity as well as increases in gill H+-ATPase protein expression level in each habitat compared with that in the common ALW treatment. Despite the presence of population-within-habitat-type differences, significant habitat-type effects were revealed in most of the ion regulation characteristics examined under different acclimation conditions. Significantly lower plasma Cl- was detected in fish from coastal rivers than in fish from the other two habitat types during the IPW treatment, which was also significantly lower compared with that in ALW. Similarly, gill Na+/K+-ATPase activity was lower in the coastal river populations in IPW than in fish from coastal and interior lakes, which was not in accordance with the protein expression in the gill. For gill H+-ATPase, fish from interior lake populations had the highest level of activity across all habitat types under all conditions, which was related to the protein levels in the gill. The activity of gill H+-ATPase was positively correlated with the combined effect of water Na+ and pH under the ALW treatment. Our results suggest that variation in habitat may be an important factor driving differences in gill Na+/K+-ATPase and H+-ATPase activity across populations of C. asper. Further, the combined effect of water Na+ and pH may have played a key role in physiological adaptation in C. asper during post-glacial freshwater colonization and dispersal.

Boosting cisplatin chemotherapy by nanomotor-enhanced tumor penetration and DNA adducts formation.

Despite many nano-based strategies devoted to delivering cisplatin for tumor therapy, its clinical benefits are compromised by poor tissue penetration and limited DNA adducts formation of the drug. Herein, a cisplatin loading nanomotor based janus structured Ag-polymer is developed for cisplatin delivery of deeper tissue and increased DNA adducts formation. The nanomotor displayed a self-propelled tumor penetration fueled by hydrogen peroxide (H2O2) in tumor tissues, which is catalytically decomposed into a large amount of oxygen bubbles by Ag nanoparticles (NPs). Notably, cisplatin could elevate the intracellular H2O2 level through cascade reactions, further promote the degradation of Ag NPs accompanied with the Ag+ release, which could downregulate intracellular Cl- through the formation of AgCl precipitate, thereby enhancing cisplatin dechlorination and Pt-DNA formation. Moreover, polymer can also inhibit the activity of ALKBH2 (a Fe2+-dependent DNA repair enzyme) by chelating intracellular Fe2+ to increase the proportion of irreparable Pt-DNA cross-links. It is found that deep tissue penetration, as well as the increased formation and maintenance of Pt-DNA adducts induced by the nanomotor afford 80% of tumor growth inhibition with negligible toxicity. This work provides an important perspective of resolving chemotherapeutic barriers for boosting cisplatin therapy.

The isosmotic point as critical salinity limit for growth and osmoregulation, but not survival, in the wolf eel Anarrhichthys ocellatus.

Members of wolf fish family Anarhichadidae have emerged as potential cold-water marine aquaculture species. This study examined growth performance and osmoregulation in juvenile wolf eel (Anarrhichthys ocellatus) held in a series of dilute salinities (30, 14, 9, and 6 ‰) over an 8-week trial. At the conclusion of the growth study, fish were sampled for analysis of gill and intestine enzyme activity, plasma ion content, and muscle moisture. Growth rate remained positive in all salinities throughout the 8-week trial. Specific growth rate was maintained above 3.0% mass day-1 at salinities of 30 and 14 ‰, but was significantly reduced at 9 (2.9% mass day-1) and 6 ‰ (2.0% mass day-1). Muscle water content increased with increasing salinity dilution (77.9% water in 30 ‰; 79.8% water in 6 ‰), and plasma osmolality (~ 320 mOsm kg-1) was maintained in salinities as dilute as 9 ‰ but was significantly lower (~ 280 mOsm kg-1) in the most dilute salinity of 6 ‰. Segmental linear regression analyses revealed that the calculated isosmotic point for wolf eel of ~ 10.6 ‰ was a critical limit for maintaining growth performance and osmoregulatory homeostasis. It is an important finding that fish considered to be a typical marine stenohaline organism could maintain ion and water balance as low as the isosmotic point, and exhibit survival and positive growth rates in salinities as dilute as 6 ‰. This work delivers a fundamental step in the empirical examination of this emerging aquaculture species and provides a model for evaluating osmoregulatory performance of marine stenohaline fishes in low-salinity aquaculture.

Shade and salinity responses of two dominant coastal wetland grasses: implications for light competition at the transition zone.

BACKGROUND: Coastal wetlands are threatened by the increased salinity that may result from sea level rise. Salinity stress alters species zonation patterns through changes in competitive outcome between species differing in salinity tolerance. This study therefore aimed to understand how salinity and light affect two dominant and competing coastal wetland grasses that differ in salt tolerance, height and photosynthetic metabolism. METHODS: The C4 species Spartina anglica and the C3 species Phragmites australis were grown at five salinity levels (0, 7, 14, 21 and 28 ppt) and two light fluxes (100 % and 50 % of natural daylight) in an outdoor experimental setup for 102 d with full access to nutrients. KEY RESULTS: Salinity reduced the biomass, height and shoot density of P. australis from 81.7 g dry weight (DW), 0.73 m and 37 shoots per pot at a salinity of 0 ppt to 16.8 gDW, 0.3 m and 14 shoots per pot at a salinity of 28 ppt. Biomass, height and shoot density of S. anglica did not respond or were only slightly reduced at the highest salinity of 28 ppt. High salinity also resulted in a higher tissue concentration of N and P in P. australis. Both species had low ability to acclimate to the lower light flux. Shade acclimation in S. anglica occurred via modest changes in specific leaf area, pigment content and biomass allocation. CONCLUSIONS: High salinity reduced traits important for light competition and increased the nutrient concentration in P. australis leaf and root biomass, while this was overall unaffected in S. anglica. This is likely to reduce the competitive ability of P. australis over S. anglica for light because at high salinities the former cannot effectively shade the lower-growing S. anglica. Neither species effectively acclimates to shade, which could explain why S. anglica does not occur in the understorey of P. australis at low salinities.

First Genome of Labyrinthula sp., an Opportunistic Seagrass Pathogen, Reveals Novel Insight into Marine Protist Phylogeny, Ecology and CAZyme Cell-Wall Degradation.

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula spp. virulence and ecology. Given these uncertainties, we investigated the Labyrinthula genus from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthula SR_Ha_C's actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialized substrate affinity and the scarcity of characterized protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula spp. ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between the Labyrinthula genus and its host substrate.

Hypoxia modifies calcium handling in the Pacific hagfish, Eptatretus stoutii.

Hagfishes may encounter low dissolved oxygen in their natural habitats, a consequence of association with hypoxic sediments and their feeding behaviour. In teleost fish, hypoxia exposure decreases ion uptake, speculated to be a mechanism for energy conservation. Although hagfishes osmoconform, they do regulate extracellular fluid concentrations of divalent cations such as calcium. The current study hypothesised that exposure of hagfish to hypoxia (0.4 kPA, 24 h) would reduce calcium uptake (determined via in vitro isolated skin and gut epithelial transport assays) and calcium accumulation (determined by in vivo whole animal exposures, using radiolabelled calcium (45Ca) to assess newly acquired calcium). A decrease in in vitro epidermal uptake was observed at sub-environmental calcium levels (10 µM), but not at environmental calcium levels (10 mM). No changes in gut calcium uptake were determined. Conversely, hypoxia led to a more rapid in vivo accumulation of calcium in tissues (skin, muscle, liver, heart, plasma, brain), mediated mostly by a significant increase in accumulation at the gill. These differences were only apparent after 1-h of exposure to the radiolabel (i.e., the last hour of the 24-h hypoxia exposure) and were not observed after 3 and 24 h periods of radiolabel exposure. This outcome was the opposite of the hypothesised effect. The reasons for a more rapid accumulation of calcium in hypoxic hagfish are unknown but may relate to roles for calcium in enhancing hypoxia tolerance in hagfishes or could be a consequence of changes in ventilatory frequency.

Examination of ionoregulatory characteristics of South American cichlids.

We examined ionoregulatory traits of four cichlid species from South America, oscars (Astronotus ocellatus), Tapajos cichlids (Geophagus sp.), Macmaster's dwarf cichlids (Apistogramma macmasteri), and keyhole cichlids (Cleithracara maronii), all inhabitants of ion-poor waters. Km values for Na+ transport in fish held in 100 µmol L-1 Na+ water ranged from 49 to 143 µmol L-1, and Jmax values spanned 450 to 1205 nmol g-1 h-1. After one month in 1000 µmol L-1 Na+ water, kinetic parameters for Na+ uptake in three of the four species acclimated, but only oscars displayed the "typical" pattern of higher Km and lower Jmax values. Low pH water inhibited Na+ uptake (JinNa) in all, and stimulated Na+ efflux (JoutNa) 2.5 to 3.5-fold in three of the four species. Oscars alone had had a measurable JinNa at pH 3.5 and no stimulation of JoutNa. We measured JinNa in oscars and keyhole cichlids during exposure to 100 µmol L-1 Ethoxzolemide (EZ), an inhibitor of carbonic anhydrase, and 1 mmol L-1 NH4Cl (HEA). EZ inhibited JinNa by about 50% and HEA reduced JinNa by 80-90%. These results suggest that Na+ uptake involves H+ extrusion. Acute exposure to 1 µmol L-1 CuSO4 and 60 nmol L-1 AgNO3 inhibited JinNa in both species by 30-85%. Exposure of oscars to 5-fold higher concentrations of both metals did not further inhibit JinNa, but it did stimulate JoutNa 50-150%. The response to metals of both species are similar to other species that have been examined.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na+/H+ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

Functional characterization and osmoregulatory role of the Na+-K+-2Cl- cotransporter in the gill of sea lamprey (Petromyzon marinus), a basal vertebrate.

The present study provides molecular and functional characterization of Na+-K+-2Cl- cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey Na-K-Cl cotransporter 1 (NKCC1), which we show groups strongly with and occupies a basal position among other vertebrate NKCC1 sequences. In postmetamorphic juvenile lamprey, nkcc1 mRNA was present in many tissues but was fivefold higher in the gill than any other examined tissue, and NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+-K+-ATPase (NKA/Atp1a1) were significantly upregulated (20- to 200-fold) during late metamorphosis in fresh water, coinciding with the development of salinity tolerance, and were upregulated an additional twofold after acclimation to seawater (SW). Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes coexpressing NKA, which develop during metamorphosis in preparation for SW entry. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality as well as reduced muscle water content after 24 h in SW; there were no effects of bumetanide in freshwater-acclimated lamprey. This work provides the first functional characterization of NKCC1 as a mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.

Expression of ion transport genes in ionocytes isolated from larval zebrafish (Danio rerio) exposed to acidic or Na+-deficient water.

In zebrafish (Danio rerio), a specific ionocyte subtype, the H+-ATPase-rich (HR) cell, is presumed to be a significant site of transepithelial Na+ uptake/acid secretion. During acclimation to environments differing in ionic composition or pH, ionic and acid-base regulations are achieved by adjustments to the activity level of HR cell ion transport proteins. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulations relied on measurements using homogenates derived from the whole body (larvae) or the gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers or transcriptional regulation specifically within individual ionocytes. The goal of the present study was to use fluorescence-activated cell sorting to separate the HR cells from other cellular subpopulations to facilitate the measurement of gene expression of HR cell-specific transporters and enzymes from larvae exposed to low pH (pH 4.0) or low Na+ (5 µM) conditions. The data demonstrate that treatment of larvae with acidic water for 4 days postfertilization caused cell-specific increases in H+-ATPase (atp6v1aa), ca17a, ca15a, nhe3b, and rhcgb mRNA in addition to increases in mRNA linked to cell proliferation. In fish exposed to low Na+, expression of nhe3b and rhcgb was increased owing to HR cell-specific regulation and elevated numbers of HR cells. Thus, the results of this study demonstrate that acclimation to low pH or low Na+ environmental conditions is facilitated by HR cell-specific transcriptional control and by HR cell proliferation.

Ionoregulatory aspects of the hypoxia-induced osmorespiratory compromise in the euryhaline Atlantic killifish (Fundulus heteroclitus): the effects of salinity.

The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas exchange and those that limit passive flux of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water flux. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In freshwater killifish, hypoxia exposure reduced branchial Na+/K+-ATPase and NEM-sensitive ATPase activities, as well as diffusive water flux rates. Unidirectional Na+ influx and Na+ efflux decreased during hypoxia in freshwater, but net Na+ flux remained unchanged. Net loss rates of Cl-, K+ and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na+ flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia to that in freshwater. In summary, freshwater and seawater killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na+ influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.

Fish embryo vulnerability to combined acidification and warming coincides with a low capacity for homeostatic regulation.

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-synthase, Na+/K+-ATPase, H+-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly hatched larvae. Treatment-related embryo mortality until hatching (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacity. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although this is probably associated with energetic trade-offs. Interestingly, whole-larvae enzyme activity (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

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.

Microplastics and copper effects on the neotropical teleost Prochilodus lineatus: Is there any interaction?

Microplastics (MP) are emerging contaminants widely found in aquatic ecosystems. In addition to MP toxicity itself, there is increasing concern about the MP adsorption capacity and the interactive effects with other contaminants, such as copper. The objective of this research was to investigate the effects of polyethylene microplastic and its association with copper (Cu) in genotoxic, biochemical, and physiological biomarkers of the neotropical teleost Prochilodus lineatus. Fish were exposed for 24 and 96 h to MP (20 µg L-1) and Cu (10 µg L-1) and MP + Cu. The results showed that MP and Cu, both isolated and in combination, promoted DNA damage in erythrocytes (96 h) and liver cells (24 and 96 h) indicating that MP and Cu are genotoxic. Fish exposed only to Cu (96 h) showed a decrease in lipid peroxidation in the liver despite of the decrease in glutathione content, indicating the efficiency of other antioxidant defenses. Brain acetylcholinesterase was inhibited in the animals from all the treatments. Although MP did not influence on Cu accumulation in tissues, decreased plasma Na+ and Ca2+ (24 h) occurred after the exposure to MP and Cu, isolated and combined. Exposure to MP and MP + Cu resulted in decreased activity of Ca2+-ATPase (24 h). Taken altogether, these results showed that MP and Cu depicted genotoxic, neurotoxic, and physiological effects on P. lineatus, both alone and combined. An interaction between Cu and MP was observed in plasma Ca2+, where the combination of both contaminants caused a greater effect than the contaminants alone.