Sub-chronic exposure to waterborne extracellular microcystin-LR impairs calcium homeostasis in rainbow trout.

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L-1 (measured values 2 and 49 µg L-1, respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L-1. A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L-1, but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown.

The Acute and Chronic Effects of a Sediment-Bound Synthetic Musk, Galaxolide, on Hyalella azteca, Chironomus dilutus, and Lumbriculus variegatus.

Galaxolide is one of the most frequently used synthetic polycyclic musks on the market and is commonly detected in aquatic waterways. Previous studies have mainly evaluated the toxicity of this emerging contaminant using water-only exposures; however, its high Log Kow (5.9) suggests that this compound is likely to partition to sediments. Three benthic invertebrates, Chironomus dilutus, Hyalella azteca, and Lumbriculus variegatus, were exposed to sediment-bound Galaxolide using both acute (10 d; survival) and chronic (28 d; survival and growth) bioassays. The acute and chronic LC50s for Galaxolide ranged from 238 to 736 mg/kg sediment (2400-7430 µg/g organic carbon [OC]) for all three species, which were above concentrations commonly detected in the environment (< 2.5 mg/kg). Growth effects (i.e., weight and/or length) were noted in two of the three organisms (with C. dilutus being the exception); however, these effects were also noted at concentrations above those that are environmentally relevant. Molecular level evaluations were conducted with surviving L. variegatus and C. dilutus collected from treatments near the LC50 value. Markers of oxidative stress (glutathione-s-transferase) and endocrine disruption (estrogen-related receptor) in C. dilutus were significantly decreased in the treatment group compared to controls by 0.7-fold and 1.9-fold, respectively. Although acute and chronic effects were largely absent at environmentally relevant concentrations, changes in endocrine response suggest that more sensitive endocrine-based endpoints, such as emergence (for C. dilutus) and molting (for H. azteca), are needed to ensure that the risk of this emerging contaminant is low at environmentally relevant concentrations.

Complex impacts of hydraulic fracturing return fluids on soil microbial community respiration, structure and functional potentials.

The consequences of soils exposed to hydraulic fracturing (HF) return fluid, often collectively termed flowback and produced water (FPW), are poorly understood, even though soils are a common receptor of FPW spills. Here, we investigate the impacts on soil microbiota exposed to FPW collected from the Montney Formation of western Canada. We measured soil respiration, microbial community structure and functional potentials under FPW exposure across a range of concentrations, exposure time and soil types (luvisol and chernozem). We find that soil type governs microbial community response upon FPW exposure. Within each soil, FPW exposure led to reduced biotic soil respiration, and shifted microbial community structure and functional potentials. We detect substantially higher species richness and more unique functional genes in FPW-exposed soils than in FPW-unexposed soils, with metagenome-assembled genomes (e.g. Marinobacter persicus) from luvisol soil exposed to concentrated FPW being most similar to genomes from HF/FPW sites. Our data demonstrate the complex impacts of microbial communities following FPW exposure and highlight the site-specific effects in evaluation of spills and agricultural reuse of FPW on the normal soil functions.

Molecular identification and postprandial regulation of glucose carrier proteins in the hindgut of Pacific hagfish, Eptatretus stoutii.

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish (Eptatretus stoutii), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[14C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.

Absence of some cytochrome P450 (CYP) and hydroxysteroid dehydrogenase (HSD) enzymes in hagfishes.

Corticosteroids are synthesized from cholesterol by steroidogenic enzyme catalysts belonging to two main families: the cytochrome p450s (CYPs) and hydroxysteroid dehydrogenases (HSDs). The action of these steroidogenic enzymes allows the genesis of the terminal active corticosteroids 11-deoxycortisol (S), 1ɑ-hydroxycorticosterone (1α-OH-B), or cortisol in different fish species. However, for Cyclostomes like hagfishes, the terminal corticosteroid is still undefined. In this study, we examined the presence or absence of CYPs and HSDs as traits in fishes to gain insight about the primary corticosteroid synthesis pathways of the hagfishes. We used published cytochrome c oxidase I (COXI) amino acid sequences to construct a phylogeny of fishes and then mapped the CYPs and HSDs as morphological traits onto the tree to predict the ancestral character states through ancestral character reconstruction (ACR). There is a clear phylogenetic signal for CYP (i.e., CYP11a1, 17, 21, and 11b) and HSD (i.e., 11-ßHSD and 3ß-HSD) derivatives of interest throughout the more derived fishes. Using trait-based ACR, we also found that hagfishes possess genes for 3ß-HSD, CYP11a1, CYP17, and CYP21. Importantly, the presence of CYP21 implies that hagfish can synthesize 11-deoxycorticosterone (11-DOC) and S. Previous research demonstrated that despite hagfish having CYP21, neither 11-DOC nor S could be detected in hagfish. This discrepancy between the presence of steroidogenic enzymes and products brings into question the expression and/or function of CYP21 in hagfishes.

Effect of amino acid composition of elastin-like polypeptide nanoparticles on nonspecific protein adsorption, macrophage cell viability and phagocytosis.

Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function.

Comparison of the Hydraulic Fracturing Water Cycle in China and North America: A Critical Review.

There is considerable debate about the sustainability of the hydraulic fracturing (HF) water cycle in North America. Recently, this debate has expanded to China, where HF activities continue to grow. Here, we provide a critical review of the HF water cycle in China, including water withdrawal practices and flowback and produced water (FPW) management and their environmental impacts, with a comprehensive comparison to the U.S. and Canada (North America). Water stress in arid regions, as well as water management challenges, FPW contamination of aquatic and soil systems, and induced seismicity are all impacts of the HF water cycle in China, the U.S., and Canada. In light of experience gained in North America, standardized practices for analyzing and reporting FPW chemistry and microbiology in China are needed to inform its efficient and safe treatment, discharge and reuse, and identification of potential contaminants. Additionally, conducting ecotoxicological studies is an essential next step to fully reveal the impacts of accidental FPW releases into aquatic and soil ecosystems in China. From a policy perspective, the development of China's unconventional resources lags behind North America's in terms of overall regulation, especially with regard to water withdrawal, FPW management, and routine monitoring. Our study suggests that common environmental risks exist within the world's two largest HF regions, and practices used in North America may help prevent or mitigate adverse effects in China.

Investigating the Potential Toxicity of Hydraulic Fracturing Flowback and Produced Water Spills to Aquatic Animals in Freshwater Environments: A North American Perspective.

Unconventional methods of oil and natural gas extraction have been a growing part of North America's energy sector for the past 20-30 years. Technologies such as horizontal hydraulic fracturing have facilitated the exploitation of geologic reserves that were previously resistant to standard drilling approaches. However, the environmental risks associated with hydraulic fracturing are relatively understudied. One such hazard is the wastewater by-product of hydraulic fracturing processes: flowback and produced water (FPW). During FPW production, transport, and storage, there are many potential pathways for environmental exposure. In the current review, toxicological hazards associated with FPW surface water contamination events and potential effects on freshwater biota are assessed. This review contains an extensive survey of chemicals commonly associated with FPW samples from shale formations across North America and median 50% lethal concentration values (LC50) of corresponding chemicals for many freshwater organisms. We identify the characteristics of FPW which may have the greatest potential to be drivers of toxicity to freshwater organisms. Notably, components associated with salinity, the organic fraction, and metal species are reviewed. Additionally, we examine the current state of FPW production in North America and identify the most significant obstacles impeding proper risk assessment development when environmental contamination events of this wastewater occur. Findings within this study will serve to catalyze further work on areas currently lacking in FPW research, including expanded whole effluent testing, repeated and chronic FPW exposure studies, and toxicity identification evaluations.

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.

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood.

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l-1 NH4 + . Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l-1 NH4 + . The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma.

Removal of biological effects of organic pollutants in municipal wastewater by a novel advanced oxidation system.

The Advanced Oxidation System (AOS) is a novel electrochemical advanced oxidation process that effectively removes bacterial and organic contaminants from wastewater. However, potential formation of secondary oxidative species may pose additional hazards to aquatic organisms living in the receiving water affected by the post-treatment effluent. The effect of exposure to AOS treated water, especially the potential long-term effects on aquatic organisms, requires further investigation to demonstrate both efficacy and safety of this process. To examine the potential adverse effects of AOS treated water, three aquatic species, including daphnia, zebrafish, and rainbow trout, were exposed to treated and untreated municipal wastewater effluent (MWE) spiked with one of two model organic contaminants, benzo[a]pyrene (BaP) and 17ß-estradiol (E2). The results indicated AOS treatment significantly reduced the adverse effects caused by exposure to MWE and model organic contaminants to baseline levels in daphnia (reduced fecundity), zebrafish embryo (elevated EROD activity), and rainbow trout (elevated plasma vitellogenin). The Ames test was also conducted to confirm the removal efficacy of carcinogenicity of BaP spiked in MWE. Overall, this study demonstrated that AOS treatment is a promising and environmentally friendly technology for wastewater treatment, remediation, and management.

Exposure to Hydraulic Fracturing Flowback Water Impairs Mahi-Mahi (Coryphaena hippurus) Cardiomyocyte Contractile Function and Swimming Performance.

Publicly available toxicological studies on wastewaters associated with unconventional oil and gas (UOG) activities in offshore regions are nonexistent. The current study investigated the impact of hydraulic fracturing-generated flowback water (HF-FW) on whole organism swimming performance/respiration and cardiomyocyte contractility dynamics in mahi-mahi (Coryphaena hippurus-hereafter referred to as "mahi"), an organism which inhabits marine ecosystems where offshore hydraulic fracturing activity is intensifying. Following exposure to 2.75% HF-FW for 24 h, mahi displayed significantly reduced critical swimming speeds (Ucrit) and aerobic scopes (reductions of ∼40 and 61%, respectively) compared to control fish. Additionally, cardiomyocyte exposures to the same HF-FW sample at 2% dilutions reduced a multitude of mahi sarcomere contraction properties at various stimulation frequencies compared to all other treatment groups, including an approximate 40% decrease in sarcomere contraction size and a nearly 50% reduction in sarcomere relaxation velocity compared to controls. An approximate 8-fold change in expression of the cardiac contractile regulatory gene cmlc2 was also seen in ventricles from 2.75% HF-FW-exposed mahi. These results collectively identify cardiac function as a target for HF-FW toxicity and provide some of the first published data on UOG toxicity in a marine species.

Ventilatory sensitivity to ammonia in the Pacific hagfish (Eptatretus stoutii), a representative of the oldest extant connection to the ancestral vertebrates.

Ventilatory sensitivity to ammonia occurs in teleosts, elasmobranchs and mammals. Here, we investigated whether the response is also present in hagfish. Ventilatory parameters (nostril flow, pressure amplitude, velar frequency and ventilatory index, the last representing the product of pressure amplitude and frequency), together with blood and water chemistry, were measured in hagfish exposed to either high environmental ammonia (HEA) in the external sea water or internal ammonia loading by intra-vascular injection. HEA exposure (10 mmol l-1 NH4HCO3 or 10 mmol l-1 NH4Cl) caused a persistent hyperventilation by 3 h, but further detailed analysis of the NH4HCO3 response showed that initially (within 5 min) there was a marked decrease in ventilation (80% reduction in ventilatory index and nostril flow), followed by a later 3-fold increase, by which time plasma total ammonia concentration had increased 11-fold. Thus, hyperventilation in HEA appeared to be an indirect response to internal ammonia elevation, rather than a direct response to external ammonia. HEA-mediated increases in oxygen consumption also occurred. Responses to NH4HCO3 were greater than those to NH4Cl, reflecting greater increases over time in water pH and PNH3  in the former. Hagfish also exhibited hyperventilation in response to direct injection of isotonic NH4HCO3 or NH4Cl solutions into the caudal sinus. In all cases where hyperventilation occurred, plasma total ammonia and PNH3  levels increased significantly, while blood acid-base status remained unchanged, indicating specific responses to internal ammonia elevation. The sensitivity of breathing to ammonia arose very early in vertebrate evolution.

Toxicity in aquatic model species exposed to a temporal series of three different flowback and produced water samples collected from a horizontal hydraulically fractured well.

In the present study, we compared the toxicity and associated chemical characterizations of flowback and produced water (FPW) collected from a single horizontal hydraulically fractured well at different time points during FPW production. Since few studies on whole mixture toxicity related to FPW exist, our aims were to determine both overall toxicity of the FPW mixture in a suite of organisms (Daphnia magna, Lumbriculus variegatus, Danio rerio, and Oncorhynchus mykiss) and also determine if toxicity changes depending on variation in FPW chemical properties as a function of time sampled (1.33, 72, and 228 h FPW samples collected immediately post-well production onset were analyzed in current study). FPW chemical composition was determined via quadra-pole inductively coupled plasma - mass spectrometry/mass spectrometry (ICP-MS/MS), full-scan high performance liquid chromatography/Orbitrap mass spectrometry (HPLC/Orbitrap-MS), and gas chromatography-mass spectrometry (GC-MS). We observed that FPW sampled later in the production process contained higher ion and total dissolved solids concentrations, whereas the highest concentrations of dissolved organic compounds were observed in the earliest FPW sample analyzed. Toxicity associated with FPW exposure was deemed to be species-specific to a certain extent, but general trends revealed the earliest FPW sampled contained highest toxic potential. Accordingly, we theorize that although the saline conditions of FPW are the foremost toxicological drivers to freshwater organisms, dissolved organics associated with FPW significantly contribute to the overall toxicity of exposed organisms.

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii.

This study used 3 H-L -alanyl-L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax -like) of 70 ± 3 nmol cm-2 h-1 and an affinity constant (Km -like) of 1072 ± 81 µM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl-L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.

Developmental Toxicity of the Organic Fraction from Hydraulic Fracturing Flowback and Produced Waters to Early Life Stages of Zebrafish ( Danio rerio).

Hydraulic fracturing (HF) has emerged as a major recovery method of unconventional oil and gas reservoirs and concerns have been raised regarding the environmental impact of releases of Flowback and Produced Water (FPW) to aquatic ecosystems. To investigate potential effects of HF-FPW on fish embryo development, HF-FPW samples were collected from two different wells and the organic fractions were isolated from both aqueous and particle phases to eliminate the confounding effects of high salinity. Each organic extract was characterized by non-target analysis with HPLC-Orbitrap-MS, with targeted analysis for polycyclic aromatic hydrocarbons provided as markers of petroleum-affected water. The organic profiles differed between samples, including PAHs and alkyl PAHs, and major substances identified by non-target analysis included polyethylene glycols, alkyl ethoxylates, octylphenol ethoxylates, and other high molecular weight (C49-79) ethylene oxide polymeric material. Zebrafish embryos were exposed to various concentrations of FPW organic extracts to investigate acute (7-day) and developmental toxicity in early life stages. The acute toxicity (LD50) of the extracted FPW fractions ranged from 2.8× to 26× the original organic content. Each extracted FPW fraction significantly increased spinal malformation, pericardial edema, and delayed hatch in exposed embryos and altered the expression of a suite of target genes related to biotransformation, oxidative stress, and endocrine-mediation in developing zebrafish embryos. These results provide novel information on the variation of organic profiles and developmental toxicity among different sources and fractions of HF-FPWs.

Functional redundancy of glucose acquisition mechanisms in the hindgut of Pacific hagfish (Eptatretus stoutii).

This study examined the mechanisms of glucose acquisition in the hindgut of Pacific hagfish (Eptatretus stoutii) using in vitro gut sac techniques. The intestine was determined to have the capacity to digest maltose into glucose along the entirety of the tract, including the foregut. Glucose uptake was biphasic and consisted of a high-affinity, low-capacity concentration-dependent component conforming to Michaelis-Menten kinetics (Km 0.37mM, Jmax 8.48nmol/cm2/h) as well as a diffusive component. There was no observed difference in glucose flux rate along the length of the intestine, similar to other nutrients investigated in the hagfish intestine. A reduced sodium (<1mM) environment did not result in a change in glucose uptake rates, likely due to a functional redundancy of glucose transporters. There was no observed effect of phloretin, yet the sodium glucose-linked transporter (SGLT)-specific inhibitor phlorizin significantly reduced glucose uptake at all concentrations tested (0.0001-1mM). Additionally, the glucose transporter (GLUT) inhibitor cytochalasin b significantly reduced glucose transport rates. The effects of these pharmacological inhibition experiments suggest the presence of multiple types of glucose transport proteins. This study clarifies the uptake strategies used by hagfish to acquire glucose at the intestine and provides insight into the evolution of such transport systems in early-diverging vertebrates.

Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish.

Na+ uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na+/H+-exchanger 3b (NHE3b) expressed in H+-ATPase-rich (HR) cells, an unidentified Na+ channel coupled to electrogenic H+-ATPase expressed in HR cells, and Na+-Cl--cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na+ channel involved in H+-ATPase-mediated Na+ uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na+ uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na+ uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na+ influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na+ uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na+ uptake, even under conditions known to constrain Na+ uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na+ uptake in larval zebrafish.

A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas).

Estuaries are environments enriched with dissolved nutrients such as amino acids. To date, marine arthropods are the only invertebrate group that have not been demonstrated to access this potentially important nutrient resource. Using in vitro gill perfusion techniques, we sought to investigate the ability of the green shore crab (Carcinus maenas) to take up the amino acid l-leucine directly from the water. Investigation of the concentration-dependent transport kinetics of radiolabelled l-leucine showed that there are two specific transport pathways across Carcinus gills, one with high affinity and low capacity, and the other with high capacity and low affinity. Using putative competitive substrates and reduced sodium preparations, we were able to identify the putative amino acid transport system associated with high-affinity uptake. This is the first study to demonstrate the absorption of dissolved organic nutrients across the gill epithelium of a marine arthropod.

Flexible ammonia handling strategies using both cutaneous and branchial epithelia in the highly ammonia-tolerant Pacific hagfish.

Hagfish consume carrion, potentially exposing them to hypoxia, hypercapnia, and high environmental ammonia (HEA). We investigated branchial and cutaneous ammonia handling strategies by which Pacific hagfish (Eptatretus stoutii) tolerate and recover from high ammonia loading. Hagfish were exposed to HEA (20 mmol/l) for 48 h to elevate plasma total ammonia (TAmm) levels before placement into divided chambers for a 4-h recovery period in ammonia-free seawater where ammonia excretion (JAmm) was measured independently in the anterior and posterior compartments. Localized HEA exposures were also conducted by subjecting hagfish to HEA in either the anterior or posterior compartments. During recovery, HEA-exposed animals increased JAmm in both compartments, with the posterior compartment comprising ~20% of the total JAmm compared with ~11% in non-HEA-exposed fish. Plasma TAmm increased substantially when whole hagfish and the posterior regions were exposed to HEA. Alternatively, plasma TAmm did not elevate after anterior localized HEA exposure. JAmm was concentration dependent (0.05-5 mmol/l) across excised skin patches at up to eightfold greater rates than in skin sections that were excised from HEA-exposed hagfish. Skin excised from more posterior regions displayed greater JAmm than those from more anterior regions. Immunohistochemistry with hagfish-specific anti-rhesus glycoprotein type c (α-hRhcg; ammonia transporter) antibody was characterized by staining on the basal aspect of hagfish epidermis while Western blotting demonstrated greater expression of Rhcg in more posterior skin sections. We conclude that cutaneous Rhcg proteins are involved in cutaneous ammonia excretion by Pacific hagfish and that this mechanism could be particularly important during feeding.