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Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (J amm ) and urea excretion (J urea ) in FW, and following FW to artificial seawater (ASW) transfer. Rates of J amm and J urea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na + /K + -ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H + -ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na + uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.

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 (T Amm ) levels before placement into divided chambers for a 4-h recovery period in ammonia-free seawater where ammonia excretion ( J Amm ) 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 J Amm in both compartments, with the posterior compartment comprising ~20% of the total J Amm compared with ~11% in non-HEA-exposed fish. Plasma T Amm increased substantially when whole hagfish and the posterior regions were exposed to HEA. Alternatively, plasma T Amm did not elevate after anterior localized HEA exposure. J Amm 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 J Amm 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., (Copyright © 2017 the American Physiological Society.)

The pesticide 3-trifluoromethyl-4-nitrophenol (TFM) is used to control sea lamprey (Petromyzon marinus) populations in the Great Lakes through its application to nursery streams containing larval sea lampreys. TFM uncouples oxidative phosphorylation, impairing mitochondrial ATP production in sea lampreys and rainbow trout (Oncorhynchus mykiss). However, little else is known about its sub-lethal effects on non-target aquatic species. The present study tested the hypotheses that TFM exposure in hard water leads to (i) marked depletion of energy stores in metabolically active tissues (brain, muscle, kidney, liver) and (ii) disruption of active ion transport across the gill, adversely affecting electrolyte homeostasis in trout. Exposure of trout to 11.0mgl(-1) TFM (12-h LC50) led to increases in muscle TFM and TFM-glucuronide concentrations, peaking at 9h and 12h, respectively. Muscle and brain glycogen was reduced by 50%, while kidney and muscle lactate increased with TFM exposure. Kidney ATP and phosphocreatine decreased by 50% and 70%, respectively. TFM exposure caused no changes in whole body ion (Na(+), Cl(-), Ca(2+), K(+)) concentrations, gill Na(+)/K(+) ATPase activity, or unidirectional Na(+) movements across the gills. We conclude that TFM causes a mismatch between ATP supply and demand in trout, leading to increased reliance on glycolysis, but it does not have physiologically relevant effects on ion balance in hard water., (© 2013.)

Metal gill binding and toxicity can be modeled using the concentration addition model, in which the toxic unit (TU) concept is used to determine if constituent metals are acting in a strictly additive, less than, or greater than additive fashion. To test this hypothesis, rainbow trout (Oncorhynchus mykiss) were exposed to a matrix of Pb plus Cd mixtures (nominal concentrations=0.75, 1.5, 2.25, 3.0 μmol L(-1)), in the presence or absence of mainly terrigenous (allochthonous; 10 mg CL(-1)) natural organic matter (NOM), and metal-gill binding, and toxicity was quantified. Based on its greater affinity for metal-gill binding sites, Cd-gill binding was expected to exceed Pb-gill binding during metal mixture exposure, but this only occurred at the lowest metal concentrations (0.75 μmol L(-1)); at higher concentrations Pb-gill binding was greater than Cd-gill binding. These unexpected observations were because Pb and Cd likely bind to different populations of high affinity, low capacity binding sites on the gill, which was borne out in subsequent attempts to mathematically model metal-gill interactions during metal-mixture exposure. The presence of an additional low affinity, high capacity population of Pb-gill binding sites also contributed to higher Pb-gill accumulation. Metal-gill interactions were complicated by NOM, which exacerbated toxicity during Cd-only exposure despite lowering Cd-gill accumulation. NOM also promoted Cd-gill binding in the presence of low-moderate concentrations of Pb (0.75 and 1.50 μmol L(-1)). We suggest that direct interactions of Cd-NOM complexes with the gill, and increases in Cd bioavailability due to Pb outcompeting Cd for NOM-metal binding sites due to its greater affinity for such ligands, accounted for greater Cd-gill binding and toxicity. We conclude that interactions of Pb and Cd with the gill cannot be predicted using the concentration addition model, and that NOM is not universally protective against metal-gill binding and toxicity when fish are exposed to metal mixtures., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Natural organic matter (NOM) in aquatic environments reduces metal toxicity to fish by forming metal-NOM complexes, which reduce metal bioavailability, metal-gill binding and toxicity. However, differences in the chemical composition of different types of NOM (quality) could also affect metal-NOM binding and toxicity. We predicted that Cu-gill binding would vary in trout exposed to Cu in the presence of NOM of different qualities. NOM was collected from three sources: Luther Marsh (terrigenous approximately allochthonous), Bannister Lake (nominally autochthonous), and from a local sewage treatment plant (designated Preston effluent). Excitation-emission matrix spectroscopy (EEMS) revealed that terrigenous Luther Marsh NOM was primarily humic acid-like material (74%), whereas Bannister Lake and Preston sewage effluent NOM had lower humic acid-like material but greater fulvic acid-like material (30% and 50%, respectively). The specific absorption coefficient (SAC) of Luther Marsh NOM was also much higher (SAC=37.8), consistent with its darker color, compared to more autochthonous, lightly coloured Bannister Lake (SAC=12.4) NOM, and Preston effluent NOM (SAC=9.2). At low-moderate waterborne Cu (0-2,000 nmol L(-1)), all NOM isolates reduced Cu-gill accumulation by 70-90%. Surprisingly, there were no measurable differences in Cu-gill binding amongst the three NOM treatments when fish were exposed to Cu in the low-moderate range. Only at higher Cu (>2,000 nmol L(-1)) were differences observed, where terrigenous Luther Marsh and Preston effluent NOM reduced Cu-gill binding by 40-50% more than the more autochthonous Bannister Lake NOM. Although Cu-gill binding estimates using the HydroQual BLM showed similar trends, the BLM consistently underestimated Cu-gill binding. We conclude that differences in Cu toxicity at lower-moderate Cu concentrations in the presence of different types of NOM are not necessarily related to measurable differences in Cu-gill accumulation. Rather, we suggest that differences in Cu toxicity reported in the presence of different types of NOM might be explained by direct actions of NOM on the gills, which are quality dependent., (2010 Elsevier B.V. All rights reserved.)

The diversity of mechanisms of ammonia and urea excretion by the gills and other epithelia of aquatic organisms, especially fish and crustaceans, has been studied for decades. Although the decades-old dogma of ;aquatic species excrete ammonia' still explains nitrogenous waste excretion for many species, it is clear that there are many mechanistic variations on this theme. Even within species that are ammonoteles, the process is not purely ;passive', often relying on the energizing effects of proton and sodium-potassium ATPases. Within the ammonoteles, Rh (Rhesus) proteins are beginning to emerge as vital ammonia conduits. Many fishes are also known to be capable of substantial synthesis and excretion of urea as a nitrogenous waste. In such species, members of the UT family of urea transporters have been identified as important players in urea transport across the gills. This review attempts to draw together recent information to update the mechanisms of ammonia and urea transport by the gills of aquatic species. Furthermore, we point out several potentially fruitful avenues for further research.

In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes., (Copyright 2002 Wiley-Liss, Inc.)