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Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America makes use of two pesticides: 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide, which are often co-applied. Sea lamprey appear to be vulnerable to these agents resulting from a lack of detoxification responses with evidence suggesting that lampricide mixtures produce a synergistic effect. However, there is a lack of information pertaining to the physiological responses of sea lamprey to niclosamide and TFM:niclosamide mixtures. Here, we characterized the transcriptomic responses of the sea lamprey to TFM, niclosamide, and a TFM:niclosamide (1.5 %) mixture in the gill. Along with a control, larval sea lamprey were exposed to each treatment for 6 h, after which gill tissues were extracted for measuring whole-transcriptome responses using RNA sequencing. Differential gene expression patterns were summarized, which included identifying the broad roles of genes and common expression patterns among the treatments. While niclosamide treatment resulted in no differentially expressed genes, TFM- and mixture-treated fish had several differentially expressed genes that were associated with the cell cycle, DNA damage, metabolism, immune function, and detoxification. However, there was no common differential expression among treatments. For the first time, we characterized the transcriptomic response of sea lamprey to niclosamide and a TFM:niclosamide mixture and identified that these agents impact mRNA transcript abundance of genes associated with the cell cycle and cellular death, and immune function, which are likely mediated through mitochondrial dysregulation. These results may help to inform the production of more targeted and effective lampricides in sea lamprey control efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (∼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates., Competing Interests: Declaration of Competing Interest We declare no competing or financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America often relies on the application of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide mixtures to kill larval sea lamprey. Selectivity of TFM against lampreys appears to be due to differential detoxification ability in these jawless fishes compared to bony fishes, particularly teleosts. However, the proximate mechanisms of tolerance to the TFM and niclosamide mixture and the mechanisms of niclosamide toxicity on its own are poorly understood, especially among non-target fishes. Here, we used RNA sequencing to identify specific mRNA transcripts and functional processes that responded to niclosamide or a TFM:niclosamide mixture in bluegill (Lepomis macrochirus). Bluegill were exposed to niclosamide or TFM:niclosamide mixture, along with a time-matched control group, and gill and liver tissues were sampled at 6, 12, and 24 h. We summarized the whole-transcriptome patterns through gene ontology (GO) term enrichment and through differential expression of detoxification genes. The niclosamide treatment resulted in an upregulation of several transcripts associated with detoxification (cyp, ugt, sult, gst), which may help explain the relatively high detoxification capacity in bluegill. Conversely, the TFM:niclosamide mixture resulted in an enrichment of processes related to arrested cell cycle and growth, and cell death alongside a diverse detoxification gene response. Detoxification of both lampricides likely involves the use of phase I and II biotransformation genes. Our findings strongly suggest that the unusually high tolerance of bluegill to lampricides is due to these animals having an inherently high capacity and flexible detoxification response to such compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Little is known about nitrogenous waste (N waste) handling and excretion ( J N waste ) during the complex life cycle of the sea lamprey ( Petromyzon marinus ), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion ( J amm ) and urea excretion ( J urea ) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of J N waste ( J N waste = J a mm + J u rea ) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher J amm and fivefold higher J urea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of J amm and J urea completely diverged following metamorphosis. In larvae, J amm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2 -like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of J amm and J urea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for J urea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Aim: Pacific hagfish are exceptionally tolerant to high environmental ammonia (HEA). Here, we elucidated a cellular mechanism that enables hagfish to actively excrete ammonia against steep ammonia gradients expected to be found inside a decomposing whale carcass., Methods: Hagfish were exposed to varying concentrations of HEA in the presence or absence of environmental Na + , while plasma ammonia levels were tracked. 14 C-methylammonium was used as a proxy for NH 4 + to measure efflux in whole animals and in isolated gill pouches; the latter allowed us to assess the effects of amiloride specifically on Na + /H + exchangers (NHEs) in gill cells. Western blotting and immunohistochemistry were utilized to evaluate the abundance and sub-cellular localization of Rhesus glycoprotein (Rh) channels in the response to HEA., Results: Hagfish actively excreted NH 4 + against steep inwardly directed E NH4 + (ΔE NH4 +  ~ 35 mV) and pNH 3 (ΔpNH 3  ~ 2000 μtorr) gradients. Active NH 4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na + , indicating a Na + /NH 4 + exchange mechanism. Active NH 4 + excretion across isolated gill pouches was amiloride-sensitive. Exposure to HEA resulted in decreased abundance of Rh channels in the apical membrane of gill ionocytes., Conclusions: During HEA exposure, hagfish can actively excrete ammonia against a steep concentration gradient using apical NHEs energized by Na + -K + -ATPase in gill ionocytes. Additionally, apical Rh channels are removed from the apical membrane, presumably to reduce ammonia loading from the environment. We suggest that this mechanism allows hagfish to maintain tolerable ammonia levels while feeding inside decomposing carrion, allowing them to exploit nutrient-rich food-falls., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Pesticides are critical for invasive species management but often have negative effects on nontarget native biota. Tolerance to pesticides should have an evolutionary basis, but this is poorly understood. Invasive sea lamprey ( Petromyzon marinus ) populations in North America have been controlled with a pesticide lethal to them at lower concentrations than native fishes. We addressed how interspecific variation in gene expression and detoxification gene diversity confer differential pesticide sensitivity in two fish species. We exposed sea lamprey and bluegill ( Lepomis macrochirus ), a tolerant native species, to 3-trifluoromethyl-4-nitrophenol (TFM), a pesticide commonly used in sea lamprey control. We then used whole-transcriptome sequencing of gill and liver to characterize the cellular response in both species. Comparatively, bluegill exhibited a larger number of detoxification genes expressed and a larger number of responsive transcripts overall, which likely contributes to greater tolerance to TFM. Understanding the genetic and physiological basis for pesticide tolerance is crucial for managing invasive species.

Invasive sea lampreys in the Laurentian Great Lakes are controlled by applying TFM (3-trifluoromethyl-4-nitrophenol) and niclosamide to streams infested with their larvae. Both agents uncouple oxidative phosphorylation in the mitochondria, but TFM specifically targets lampreys, which have a lower capacity to detoxify the lampricide. Niclosamide lacks specificity and is more potent than TFM. However, its greater potency is poorly understood. We tested the hypothesis that niclosamide is a stronger uncoupler of mitochondrial oxidative phosphorylation than TFM by measuring oxygen consumption rates in isolated liver mitochondria exposed to physiologically relevant concentrations of TFM, niclosamide, or their mixture (100 TFM:1 niclosamide) at environmentally relevant temperatures (7, 13, and 25 °C). Niclosamide increased State 4 respiration and decreased the respiratory control ratio (RCR) at much lower concentrations than TFM. Calculations of the relative EC 50 values, the amount of TFM or niclosamide required to decrease the RCR by 50%, indicated that niclosamide was 40-60 times more potent than TFM. Warmer temperature did not appear to decrease the sensitivity of mitochondria to niclosamide or TFM, as observed in the intact sea lamprey exposed to TFM in warmer waters. We conclude that the extreme sensitivity of mitochondria to niclosamide contributes to its greater in vivo toxicity in the whole animal.

Since the 1960s, chemical control of larval sea lamprey has been achieved using the pesticides 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide (Bayluscide®). Much more potent, niclosamide is often used as an adjuvant for TFM, and on its own to treat lentic habitats, rivers with high discharge and currents, and for population surveys. Yet, little is known about its mode of action or physiological effects on sea lamprey. Like TFM, niclosamide is thought to impair mitochondrial ATP production by uncoupling oxidative phosphorylation. We therefore tested the hypothesis that niclosamide would result in metabolic perturbations and disturbances to acid-base balance in larval lamprey due to their need to balance ATP supply with ATP demands. When larval sea lamprey were exposed to the nominal 9-h niclosamide LC 50 (0.11 mg L -1 ) over 9 h, it resulted in significant decreases in brain, phosphocreatine (35 %) and glycogen (50 %), accompanied by a 5-fold increase in lactate. In carcass, there were 25-30 % decreases in glycogen, corresponding increases in pyruvate and lactate, and a pronounced 0.5 unit decrease in intracellular pH. Calculation of the NAD + /NADH ratio in the carcass indicated that neither oxygen delivery nor the flux of reducing equivalents through the mitochondrial electron transport chain were impaired by niclosamide, supporting the hypothesis that niclosamide interferes with mitochondrial ATP production by uncoupling oxidative phosphorylation. Thus, greater reliance on glycogen, characterized by higher rates of glycolysis, temporarily mitigates the corresponding shortfall in ATP supply caused by niclosamide. Notably, all lamprey that survived niclosamide exposure readily restored ATP, phosphocreatine, glycogen and acid-base balance after recovery in niclosamide-free water. This resilience suggests that sea lamprey that survive or escape niclosamide treatment could compromise sea lamprey control efforts by subsequently completing their larval stage and developing into parasitic juvenile sea lamprey that could ultimately threaten Great Lake's fisheries populations., (Copyright © 2021. Published by Elsevier B.V.)

Since the 1960s, invasive sea lamprey (Petromyzon marinus) populations in the Laurentian Great Lakes have been controlled by applying two chemicals, 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide, aka. Bayluscide®), to streams infested with larval sea lamprey. These "lampricide" applications primarily rely on TFM, and are often combined with 1-2% niclosamide, which increases treatment effectiveness. Niclosamide is also used alone to treat lentic habitats and in rivers with high discharge. However, little is known about niclosamide's possible adverse physiological effects on non-target organisms. Of particular concern is the lake sturgeon (Acipenser fulvescens), which is threatened throughout the Great Lakes basin where its habitat often overlaps with larval lamprey. Because niclosamide is believed to impair ATP production by uncoupling oxidative phosphorylation, we determined how it altered metabolic processes and acid-base balance in young-of-the-year (YOY) lake sturgeon exposed to their 9-h LC 50 of niclosamide (0.11 mg L -1 ) for 9 h. Exposure to niclosamide led to decreased brain ATP and glucose reserves, and increased lactate, with no effect on brain glycogen. In contrast, substantial (60%) reductions in glycogen were observed in liver, suggesting that hepatic glycogen reserves were mobilized to meet the brain's glucose requirements when ATP supply was impaired during niclosamide exposure. Disturbances in carcass included reduced phosphocreatine (65-70%), 2- and 4-fold increases in pyruvate and lactate, and a slight metabolic acidosis, characterized by a 0.1 unit decrease in intracellular pH (pHi). Each of these disturbances were corrected within 24 h following depuration in clean (niclosamide-free) water. We conclude that if lake sturgeon survive exposure to niclosamide, they are able to rapidly replenish their energy stores (glycogen, ATP, phosphocreatine) and correct any corresponding metabolic disturbances within 24 h., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The pesticide 3-trifluoromethyl-4-nitrophenol (TFM) is applied to rivers and streams draining into the Laurentian Great Lakes to control populations of invasive sea lamprey ( Petromyzon marinus ), which are ongoing threats to fisheries during the lamprey's hematophagous, parasitic juvenile life stage. While TFM targets larval sea lamprey during treatments, threatened populations of juvenile lake sturgeon ( Acipenser fulvescens ), particularly young-of-the-year (<100 mm in length), may be adversely affected by TFM when their habitats overlap with larval sea lamprey. Exposure to TFM causes marked reductions in tissue glycogen and high energy phosphagens in lamprey and rainbow trout ( Oncorhynchus mykiss ) by interfering with oxidative ATP production in the mitochondria. To test that environmentally relevant concentrations of TFM would similarly affect juvenile lake sturgeon, we exposed them to the larval sea lamprey minimum lethal concentration (9-h LC 99.9 ), which mimicked concentrations of a typical lampricide application and quantified energy stores and metabolites in the carcass, liver and brain. Exposure to TFM reduced brain ATP, PCr and glycogen by 50-60%, while lactate increased by 45-50% at 6 and 9 h. A rapid and sustained depletion of liver glucose and glycogen of more than 50% was also observed, whereas the respective concentrations of ATP and glycogen were reduced by 60% and 80% after 9 h, along with higher lactate and a slight metabolic acidosis (~0.1 pH unit). We conclude that exposure to environmentally relevant concentrations of TFM causes metabolic disturbances in lake sturgeon that can lead to impaired physiological performance and, in some cases, mortality. Our observations support practices such as delaying TFM treatments to late summer/fall or using alternative TFM application strategies to mitigate non-target effects in waters where lake sturgeon are present. These actions would help to conserve this historically and culturally significant species in the Great Lakes., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

Control of invasive sea lamprey (Petromyzon marinus) in the Laurentian Great Lakes of North America uses lampricides, which consist of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide. Lampricides are thought to inhibit aerobic energy synthesis, with TFM having a relatively greater selective action against lampreys. While the toxicity and physiological effects of TFM are known, the impacts associated with exposure to niclosamide and TFM:niclosamide mixtures are poorly characterized in fishes. Therefore, focusing on energy metabolism, we quantified the physiological responses of larval sea lamprey and bluegill (Lepomis macrochirus), a non-target, native species. Exposures consisted of each lampricide alone (TFM at the species-specific 24 h LC 10 ; niclosamide at 1.5% of the mixture's TFM concentration) or a mixture of the two (larval sea lamprey at TFM 24 h LC 10  + 1.5% niclosamide; bluegill at sea lamprey's TFM 24 h LC 99. 9  + 1.5% niclosamide) for 24 h. Tissues (brain, skeletal muscle, and liver) were sampled at 6, 12, and 24 h of exposure and assayed for concentrations of ATP, phosphocreatine, glycogen, lactate, and glucose and tissue lampricide levels. In larval sea lamprey, TFM had little effect on brain and skeletal muscle, but niclosamide resulted in a depletion of high energy substrates in both tissues. Mixture-exposed lamprey showed depletion of high energy substrates, accumulation of lactate, and high mortality rates. Bluegill were largely unaffected by toxicant exposures. However, bluegill liver showed lower glycogen and lactate under all three toxicant exposures suggesting increased metabolic turnover. Bluegill also had lower concentrations of TFM and niclosamide in their tissues when compared to lamprey. Our results indicate that lampricide toxicity in sea lamprey larvae is mediated through a depletion of high energy substrates because of impaired aerobic ATP synthesis. We also confirmed that non-target bluegill showed high tolerance to lampricide exposure, an effect potentially mediated through a high detoxification capacity relative to lampreys., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The toxic unit and additive index approaches were used to understand how 2 pesticides, 3-trifluoromethyl-4-nitrophenol (TFM) and 2,5-dichloro-4-nitrosalicylanilide (niclosamide; Nic), interact in mixtures. Our first objective was to determine whether the interaction was strictly additive or greater than additive at doses comparable to those used to control invasive sea lamprey (Petromyzon marinus) in the Laurentian Great Lakes, and our second was to compare the utility of the toxic unit and additive index models for determining how TFM and Nic interacted. Typically, TFM is mixed with Nic (1-2%, w/v) to increase its potency and reduce TFM use. However, there is little information on how the 2 chemicals interact. Using a well-studied, resident nontarget fish, the rainbow trout (Oncorhynchus mykiss), we conducted toxicity tests with TFM, Nic, and TFM:Nic (100:1, w/v; TFM/1% Nic) mixtures over 12 h to determine if the interaction was strictly additive, less than additive (antagonistic), or greater than additive (synergistic). The toxic unit and additive index approaches indicated synergistic interactions at environmentally relevant concentrations, suggesting that both are valid approaches for predicting how TFM and Nic interact. The toxic unit approach was simpler to conceptualize and to calculate, and we recommend that it be used when describing how TFM and Nic, and other similar organic compounds, interact with each other in aquatic ecosystems. Environ Toxicol Chem 2021;40:1419-1430. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

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.

Control programs are implemented to mitigate the damage caused by invasive species worldwide. In the highly invaded Great Lakes, the climate is expected to become warmer with more extreme weather and variable precipitation, resulting in shorter iced-over periods and variable tributary flows as well as changes to pH and river hydrology and hydrogeomorphology. We review how climate change influences physiology, behavior, and demography of a damaging invasive species, sea lamprey (Petromyzon marinus), in the Great Lakes, and the consequences for sea lamprey control efforts. Sea lamprey control relies on surveys to monitor abundance of larval sea lamprey in Great Lakes tributaries. The abundance of parasitic, juvenile sea lampreys in the lakes is calculated by surveying wounding rates on lake trout (Salvelinus namaycush), and trap surveys are used to enumerate adult spawning runs. Chemical control using lampricides (i.e., lamprey pesticides) to target larval sea lamprey and barriers to prevent adult lamprey from reaching spawning grounds are the most important tools used for sea lamprey population control. We describe how climate change could affect larval survival in rivers, growth and maturation in lakes, phenology and the spawning migration as adults return to rivers, and the overall abundance and distribution of sea lamprey in the Great Lakes. Our review suggests that Great Lakes sea lamprey may benefit from climate change with longer growing seasons, more rapid growth, and greater access to spawning habitat, but uncertainties remain about the future availability and suitability of larval habitats. Consideration of the biology of invasive species and adaptation of the timing, intensity, and frequency of control efforts is critical to the management of biological invasions in a changing world, such as sea lamprey in the Great Lakes., (© 2019 John Wiley & Sons Ltd.)

The role of lamprey epithelium tight junctions (TJs) in the regulation of salt and water balance is poorly understood. This study reported on claudin (Cldn) TJ protein transcripts of pre-metamorphic larval and post-metamorphic juvenile sea lamprey (Petromyzon marinus) and the transcriptional response of genes encoding Cldns to changed environmental ion levels. Transcripts encoding Cldn-3b, -4, -5, -10, -14, -18 and -19 were identified, and mRNA expression profiles revealed the organ-specific presence of cldn-5 and -14, broad expression of cldn-3b, -4, -10, -18 and -19 and spatial differences in the mRNA abundance of cldn-4, -3b and -14 along the ammocoete intestine. Expression profiles were qualitatively similar in ammocoetes and juvenile fishes. Transcript abundance of genes encoding Cldns in osmoregulatory organs (gill, kidney, intestine and skin) was subsequently investigated after exposure of ammocoetes to ion-poor water (IPW) and juveniles to hyperosmotic conditions [60% sea water (SW)]. IPW-acclimated ammocoetes increased mRNA abundance of nearly all cldns in the gill. Simultaneously, cldn-10 abundance increased in the skin, whereas cldn-4, -14 and -18 decreased in the kidney. Ammocoete cldn mRNA abundance in the intestine was altered in a region-specific manner. In contrast, cldn transcript abundance was mostly downregulated in osmoregulatory organs of juvenile fish acclimated to SW - cldn-3b, -10 and -19 in the gill; cldn-3b, -4, -10 and -19 in the skin; cldn-3b in the kidney; and cldn-3b and -14 in the intestine. Data support the idea that Cldn TJ proteins play an important role in the osmoregulatory physiology of pre- and post-metamorphic sea lamprey and that Cldn participation can occur across organs, in an organ-specific manner, as well as differ spatially within organs, which contributes to the regulation of salt and water balance in these fishes., (© 2020 The Fisheries Society of the British Isles.)

The pesticide, 3-trifluoromethyl-4-nitrophenol (TFM), is used to control invasive sea lamprey ( Petromyzon marinus ) populations in the Laurentian Great Lakes. Applied to infested tributaries, it is most toxic to larval sea lamprey, which have a low capacity to detoxify TFM. However, TFM can be toxic to lake sturgeon ( Acipenser fulvescens ), whose populations are at risk throughout the basin. They are most vulnerable to TFM in early life stages, with the greatest risk of non-target mortality occurring in waters with high alkalinity. We quantified TFM toxicity and used radio-labelled TFM ( 14 C-TFM) to measure TFM uptake rates in lake sturgeon in waters of different pH and alkalinity. Regardless of pH or alkalinity, TFM uptake was 2-3-fold higher in young-of-the-year (YOY) than in age 1-year-plus (1+) sturgeon, likely due to higher mass-specific metabolic rates in the smaller YOY fish. As expected, TFM uptake was highest at lower (pH 6.5) versus higher (pH 9.0) pH, indicating that it is taken up across the gills by diffusion in its unionized form. Uptake decreased as alkalinity increased from low (~50 mg L -1 as CaCO 3 ) to moderate alkalinity (~150 mg L -1 as CaCO 3 ), before plateauing at high alkalinity (~250 mg L -1 as CaCO 3 ). Toxicity curves revealed that the 12-h LC 50 and 12-h LC 99.9 of TFM to lake sturgeon were in fact higher (less toxic) than in sea lamprey, regardless of alkalinity. However, in actual treatments, 1.3-1.5 times the minimum lethal TFM concentration (MLC = LC 99.9 ) to lamprey is applied to maximize mortality, disproportionately amplifying TFM toxicity to sturgeon at higher alkalinities. We conclude that limiting TFM treatments to late summer/early fall in waters of moderate-high alkalinity, when lake sturgeon are larger with lower rates of TFM uptake, would mitigate non-target TFM effects and help conserve populations of these ancient, culturally important fishes., (© The Author(s) 2019. Published by Oxford University Press and the Society for Experimental Biology.)

The invasion of the Laurentian Great Lakes of North America by sea lampreys (Petromyzon marinus) in the early 20th century contributed to the depletion of commercial, recreational and culturally important fish populations, devastating the economies of communities that relied on the fishery. Sea lamprey populations were subsequently controlled using an aggressive integrated pest-management program which employed barriers and traps to prevent sea lamprey from migrating to their spawning grounds and the use of the piscicides (lampricides) 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide to eliminate larval sea lampreys from their nursery streams. Although sea lampreys have not been eradicated from the Great Lakes, populations have been suppressed to less than 10% of their peak numbers in the mid-1900s. The ongoing use of lampricides provides the foundation for sea lamprey control in the Great Lakes, one of the most successful invasive species control programs in the world. Yet, significant gaps remain in our understanding of how lampricides are taken-up and handled by sea lampreys, how lampricides exert their toxic effects, and how they adversely affect non-target invertebrate and vertebrates species. In this review we examine what has been learned about the uptake, handling and elimination, and the mode of TFM and niclosamide toxicity in lampreys and in non-target animals, particularly in the last 10 years. It is now clear that the mode of TFM toxicity is the same in non-target fishes and lampreys, in which TFM interferes with oxidative phosphorylation by the mitochondria leading to decreased ATP production. Vulnerability to TFM is related to abiotic factors such as water pH and alkalinity, which we propose changes the relative amounts of the bioavailable un-ionized form of TFM in the gill microenvironment. Niclosamide, which is also a molluscicide used to control snails in areas prone to schistosomiasis infections of humans, also likely works by uncoupling oxidative phosphorylation, but less is known about other aspects of its toxicology. The effects of TFM include reductions in energy stores, particularly glycogen and high energy phosphagens. However, non-target fishes readily recover from sub-lethal TFM exposure as demonstrated by the rapid restoration of energy stores and clearance of TFM. Although both TFM and niclosamide are non-persistent in the environment and critical for sea lamprey control, increasing public and institutional concerns about pesticides in the environment makes it imperative to explore other means of sea lamprey control. Accordingly, we also address possible "next-generation" strategies of sea lamprey control including genetic tools such as RNA interference and CRISPR-Cas9 to impair critical physiological processes (e.g. reproduction, digestion, metamorphosis) in lamprey, and the use of green chemistry to develop more environmentally benign chemical methods of sea lamprey control., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Lampricides are currently being applied to streams and rivers to control the population of sea lamprey, an invasive species, in the Great Lakes. The most commonly used lampricide agent used in the field is 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey in lamprey-infested rivers and streams. The specificity of TFM is due to the relative inability of sea lamprey to detoxify the agent relative to non-target fishes. There is increasing concern, however, about non-target effects on fishes, particularly threatened populations of juvenile lake sturgeon (LS; Acipenser fulvescens). There is therefore a need to develop models to better define lake sturgeon's response to TFM. Here we report the establishment of five LS cell lines derived from the liver, gill, skin and intestinal tract of juvenile LS and some of their cellular characteristics. All LS cell lines grew well at 25 °C in Leibovitz's (L)- 15 medium supplemented with 10% FBS. All cell lines demonstrated high senescence-associated β-galactosidase activity and varying levels of Periodic acid Schiff-positive polysaccharides, indicating substantial production of glycoproteins and mucosubstances by the cells. Comparative toxicity of TFM in the five LS cell lines was assessed by two fluorescent cell viability dyes, Alamar Blue and CFDA-AM, in conditions with and without serum and at 24 or 72 h exposure. Deduced EC 50 values were compared between the cell lines and to the reported in vivo LC 50 s. Tissues sensitive to the effects of TFM in vivo correlated with cell lines from the same tissues being most sensitive to TFM in vitro. EC 50 values for the LSliver-e cells was significantly lower than the EC 50 for the rainbow trout (RBT) liver cells RTL-W1, reaffirming the in vivo observation that LS was generally more TFM-sensitive than rainbow trout. Our data suggests that whole-fish sensitivity of LS to TFM is likely attributable to sensitivity at the cellular level. Thus, LS cell lines, as well as those of RBT, can be used to screen and evaluate the toxicity of the next generation of lampricides on non-target fish such as lake sturgeon., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The piscicide 3-trifluoromethyl-4-nitrophenol (TFM) has been used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes for almost 60 years. Applied to rivers and streams containing larval lampreys, TFM seldom harms non-target fishes, but the effects of sub-lethal treatments on fish physiology are not well understood. We examined the effects of 9 h exposure to TFM on the stress axis and liver metabolic capacity of rainbow trout (Oncorhynchus mykiss) using in vivo and in vitro approaches. The fish that had been acutely exposed to TFM in vivo had increased plasma cortisol levels at 12 h post-treatment, but TFM exposure did not interfere with in vitro cortisol production in head kidney preparations. Subjecting trout to an acute handling stressor 12 h post-TFM exposure resulted in a relative attenuation of the plasma cortisol and glucose response compared to pre-stress levels. We conclude that routine TFM treatments can lead to elevations of plasma cortisol following exposure, plus a relative dampening of the stress response in rainbow trout, with high cortisol levels lasting at least 12 h post-treatment. Since the ability of the fish to produce cortisol and the liver metabolic capacity were not compromised following TFM exposure, it is likely that their ability to cope with other stressors is not altered in the long-term., Competing Interests: The authors have declared that no competing interests exist.

Invasive sea lamprey (Petromyzon marinus) are controlled in the Great Lakes using the lampricide 3-trifluoromethyl-4-nitrophenol (TFM), which is applied to streams infested with larval lamprey. However, lamprey that survive treatments (residuals) remain a challenge because they may subsequently undergo metamorphosis into parasitic juvenile animals that migrate downstream to the Great Lakes, where they feed on important sport and commercial fishes. The goal of this study was to determine if body size and life stage could potentially influence sea lamprey tolerance to TFM by influencing patterns of TFM uptake and elimination. Because mass specific rates of oxygen consumption (M˙O 2 ) are lower in larger compared to smaller lamprey, we predicted that TFM uptake would be negatively correlated to body size, suggesting that large larvae would be more tolerant to TFM exposure. Accordingly, TFM uptake and M˙O 2 were measured in larvae ranging in size from 0.2-4.2g using radio-labelled TFM ( 14 C-TFM) and static respirometry. Both were inversely proportional to wet mass (M), and could be described usingthe allometric power relationship: Y=aM b , in which M˙O 2 =1.86M 0.53 and TFM Uptake=7.24M 0.34 . We also predicted that body size would extend to rates of TFM elimination, which was measured following the administration of 14 C-TFM (via intraperitoneal injection). However, there were no differences in the half-lives of elimination of TFM (T 1/2 -TFM). There were also no differences in M˙O 2 or TFM uptake amongst size-matched larval, metamorphosing (stages 6-7), or post-metamorphic (juvenile) sea lamprey. However, the T 1/2 -TFM was significantly lower in larval than post-metamorphic lamprey (juvenile), indicating the larval lamprey cleared TFM more efficiently than juvenile lamprey. We conclude that larger larval sea lamprey are more likely to survive TFM treatments suggesting that body size might be an important variable to consider when treating streams with TFM to control these invasive species., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

This study reports on tight junction-associated MARVEL proteins of larval sea lamprey ( Petromyzon marinus ) and their potential role in ammocoete osmoregulation. Two occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln , ocln-a and tric were broadly expressed in larval lamprey, with the greatest abundance of ocln in the gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins, respectively, while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na + ] and [Cl - ] decreased, but not [Ca 2+ ], and carcass moisture content increased. In association, Ocln abundance increased in the skin and kidney, but reduced in the gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln , ocln-a and tric mRNA abundance were also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes are controlled by applying the piscicide, 3-trifluoromethyl-4-nitrophenol (TFM), to infested streams with larval sea lamprey (ammocoetes). While treatment mortality is >90%, surviving lamprey, called residuals, can undermine control efforts. A key determinant of TFM effectiveness is water pH, which can fluctuate daily and seasonally in surface waters. The objectives of this research were to evaluate the influence of pH on the uptake, elimination, and accumulation of TFM by larval sea lamprey using radio-labeled TFM ( 14 C-TFM), when exposed to a nominal concentration of 4.6mgTFML -1 or 7.6mgTFML -1 , 3h or 1h, respectively. TFM uptake rates were approximately 5.5-fold greater at low pH (6.86) compared to the high pH (8.78), most likely due to the unionized, lipophilic form of TFM existing in greater amounts at a lower pH. In contrast, elimination rates following the injection of 85nmolTFMg -1 body mass were 1.7-1.8 fold greater at pH8.96 than at pH6.43 during 2-4h of depuration in TFM-free water. Greater initial excretion rates at pH8.96 were presumably due to predicted increases in outward concentration gradients of un-ionized TFM. The present findings suggest that TFM is mainly taken-up in its un-ionized form, more lipophilic form, but there is also significant uptake of the ionized form of TFM via an unknown mechanism. Moreover, we provide an explanation to how small increases in pH can undermine lampricide treatment success increasing residual lamprey populations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

With oxygen deprivation, the mammalian brain undergoes hyper-activity and neuronal death while this does not occur in the anoxia-tolerant goldfish ( Carassius auratus ). Anoxic survival of the goldfish may rely on neuromodulatory mechanisms to suppress neuronal hyper-excitability. As γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, we decided to investigate its potential role in suppressing the electrical activity of goldfish telencephalic neurons. Utilizing whole-cell patch-clamp recording, we recorded the electrical activities of both excitatory (pyramidal) and inhibitory (stellate) neurons. With anoxia, membrane potential ( V m ) depolarized in both cell types from -72.2 mV to -57.7 mV and from -64.5 mV to -46.8 mV in pyramidal and stellate neurons, respectively. While pyramidal cells remained mostly quiescent, action potential frequency (AP f ) of the stellate neurons increased 68-fold. Furthermore, the GABA A receptor reversal potential ( E - GABA ) was determined using the gramicidin perforated-patch-clamp method and found to be depolarizing in pyramidal (-53.8 mV) and stellate neurons (-42.1 mV). Although GABA was depolarizing, pyramidal neurons remained quiescent as E GABA was below the action potential threshold (-36 mV pyramidal and -38 mV stellate neurons). Inhibition of GABA A receptors with gabazine reversed the anoxia-mediated response. While GABA B receptor inhibition alone did not affect the anoxic response, co-antagonism of GABA A and GABA B receptors (gabazine and CGP-55848) led to the generation of seizure-like activities in both neuron types. We conclude that with anoxia, V m depolarizes towards E GABA which increases AP f in stellate neurons and decreases AP f in pyramidal neurons, and that GABA plays an important role in the anoxia tolerance of goldfish brain., (© 2017. Published by The Company of Biologists Ltd.)

Buildups of ammonia can cause potentially fatal brain swelling in mammals, but such swelling is reversible in the anoxia- and ammonia-tolerant goldfish (Carassius auratus). We investigated brain swelling and its possible relationship to oxidative stress in the brain and liver of goldfish acutely exposed to high external ammonia (HEA; 5 mmol/l NH 4 Cl) at two different acclimation temperatures (14°C, 4°C). Exposure to HEA at 14°C for 72h resulted in increased internal ammonia and glutamine concentrations in the brain, and it caused cellular oxidative damage in the brain and liver. However, oxidative damage was most pronounced in brain, in which there was a twofold increase in thiobarbituric acid-reactive substances, a threefold increase in protein carbonylation, and a 20% increase in water volume (indicative of brain swelling). Increased activities of catalase, glutathione peroxidase, and glutathione reductase in the brain suggested that goldfish upregulate their antioxidant capacity to partially offset oxidative stress during hyperammonemia at 14°C. Notably, acclimation to colder (4°C) water completely attenuated the oxidative stress response to HEA in both tissues, and there was no change in brain water volume despite similar increases in internal ammonia. We suggest that ammonia-induced oxidative stress may be responsible for the swelling of goldfish brain during HEA, but further studies are needed to establish a mechanistic link between reactive oxygen species production and brain swelling. Nevertheless, a high capacity to withstand oxidative stress in response to variations in internal ammonia likely explains why goldfish are more resilient to this stressor than most other vertebrates., (Copyright © 2017 the American Physiological Society.)

Increased internal ammonia (hyperammonemia) and ischemic/anoxic insults are known to result in a cascade of deleterious events that can culminate in potentially fatal brain swelling in mammals. It is less clear, however, if the brains of fishes respond to ammonia in a similar manner. The present study demonstrated that the crucian carp (Carassius carassius) was not only able to endure high environmental ammonia exposure (HEA; 2 to 22 mmol L(-1)) but that they experienced 30% increases in brain water content at the highest ammonia concentrations. This swelling was accompanied by 4-fold increases in plasma total ammonia (TAmm) concentration, but both plasma TAmm and brain water content were restored to pre-exposure levels following depuration in ammonia-free water. The closely related, ammonia-tolerant goldfish (Carassius auratus) responded similarly to HEA (up to 3.6 mmol L(-1)), which was accompanied by 4-fold increases in brain glutamine. Subsequent administration of the glutamine synthetase inhibitor, methionine sulfoximine (MSO), reduced brain glutamine accumulation by 80% during HEA. However, MSO failed to prevent ammonia-induced increases in brain water content suggesting that glutamine may not be directly involved in initiating ammonia-induced brain swelling in fishes. Although the mechanisms of brain swelling are likely different, exposure to anoxia for 96 h caused similar, but lesser (10%) increases in brain water content in crucian carp. We conclude that brain swelling in some fishes may be a common response to increased internal ammonia or lower oxygen but further research is needed to deduce the underlying mechanisms behind such responses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Pacific hagfish (Eptatretus stoutii) has an exceptional ability to both withstand and recover from exposure to high external ammonia (HEA). This tolerance is likely due to the feeding behavior of this scavenger, which feeds on intermittent food falls of carrion (e.g. fish, large marine mammals) during which time it may be exposed to high concentrations of total ammonia (T(Amm)=NH3+NH4(+)) while burrowed inside the decomposing carcass. Here we exposed hagfish to 20 mmol L(-1) T(Amm) for periods of up to 48 h and then let animals recover in ammonia-free seawater. During the 48 h HEA exposure period, plasma T(Amm) increased 100-fold to over 5000 μmol L(-1) while ammonia excretion (J(amm)) was transiently inhibited. This increase in plasma T(Amm) resulted from NH3 influx down massive inwardly directed ΔP(NH3) gradients, which also led to a short-lived metabolic alkalosis. Plasma [T(Amm)] stabilized after 24-48 h, possibly through a reduction in NH3 permeability across the body surface, which lowered NH3 influx. Ammonia balance was subsequently maintained through the re-establishment of J(amm) against an inwardly directed ΔP(NH3). Calculations of the Nernst potential for ammonia strongly indicated that J(amm) was also taking place against a large inwardly directed NH4(+) electrochemical gradient. Recovery from HEA in ammonia-free water was characterized by a large ammonia washout, and the restoration of plasma TAmm concentrations to near control concentrations. Ammonia clearance was also accompanied by a residual metabolic acidosis, which likely offset the ammonia-induced metabolic alkalosis seen in the early stages of HEA exposure. We conclude that restoration of J(amm) by the Pacific hagfish during ammonia exposure likely involves secondary active transport of NH4(+), possibly mediated by Na(+)/NH4(+) (H(+)) exchange., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The physiological and toxicological effects of Cd and Pb have been thoroughly studied, but relatively little work has been done to determine how mixtures of these metals affect fishes in soft (<100 μmol L(-1)Ca(2+)) slightly acidic (pH ∼6) waters typical of many lakes in the Canadian Shield and other regions. Recently, it has been suggested that acute exposure to Cd plus Pb mixtures (3h) had greater than additive effects on both Ca(2+) and Na(+) influx, which could potentially exacerbate disturbances to ion balance and result in greater toxicity in rainbow trout (Oncorhynchus mykiss). The goal of the present study was to test this hypothesis by assessing the physiological and toxicological effects of Cd plus Pb mixtures over longer time periods (3-5 days), but at relatively low, more environmentally relevant concentrations of these metals. Accordingly, toxicity and measurements of blood acid-base regulation (PaO2, pHa), hematology (Ht, Hb, MCHC, and Protein), ionic composition (body ions and plasma Ca(2+), Na(+), Cl(-), osmolality), unidirectional Na(+) fluxes and branchial Na(+)/K(+)-ATPase activity were measured in rainbow trout exposed to Cd plus Pb mixtures. Experiments on rainbow trout, implanted with dorsal aortic catheters for repetitive blood sampling, demonstrated that exposure to Pb alone (26 nmol PbL(-1)) was less toxic than Cd alone (6 nmol CdL(-1)), which was much less toxic to the fish than a Cd plus Pb mixture (7 nmol CdL(-1) plus 45 nmol PbL(-1)), which led to greater than additive 80% mortality by 5d. Both Cd and Pb inhibited Na(+) influx over 3d exposure to the metals, which was partially offset by decreases in the diffusive efflux (outflux) of Na(+) across the gill. Despite an absence of detectable effects of Pb alone on plasma ion balance, Cd plus Pb mixtures exacerbated Cd-induced reductions in plasma Ca(2+) concentration, and resulted in pronounced reductions in plasma Na(+), Cl(-), and osmolality. No effects on Na(+)/K(+)-ATPase activity were noted following exposure to Cd, Pb or Pb plus Cd mixtures. We conclude that the greater than additive toxicity of Cd plus Pb mixtures observed in the present and previous studies is because these metals not only have common, but also independent binding sites and mechanisms of action, which could exacerbate the pathophysiological effects caused by each metal alone., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The primary method of sea lamprey (Petromyzon marinus) control in the Great Lakes is the treatment of streams and rivers with the pesticide 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey. However, less is known about the effects of TFM on other stages of the sea lamprey's complex life cycle. The goal of this study was to determine how TFM affected internal energy stores, metabolites, and ion balance in larval, juvenile (parasitic) and adult sea lamprey. The larvae were more tolerant to TFM than the adults, with a 2-fold higher 12h TFM LC50 and a 1.5-fold higher LC99.9. Acute (3h) exposure of the larvae, parasites and adults to their respective 12h TFM LC99.9 led to marked reductions in glycogen and phosphocreatine in the adult brain, with lesser or no effect in the larvae and parasites. Increased lactate in the brain, at less than the expected stoichiometry, suggested that it was exported to the blood. Kidney glycogen declined after TFM exposure, suggesting that this organ plays an important role in glucose homeostasis. TFM-induced disturbances to ion balance were minimal. In conclusion, TFM perturbs energy metabolism in all major stages of the sea lamprey life cycle in a similar fashion, but the adults appear to be the most sensitive. Thus, the adult stage could be a viable and effective target for TFM treatment, particularly when used in combination with other existing and emerging strategies of sea lamprey control., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.)

In higher vertebrates, phagocytosis plays a critical role in development and immunity, based on the internalization and removal of apoptotic cells and invading pathogens, respectively. Previous studies describe the effective uptake of these particles by lower vertebrate and invertebrate phagocytes, and identify important molecular players that contribute to this internalization. However, it remains unclear if individual phagocytes mediate internalization processes in these ancient organisms, and how this impacts the balance of pro-inflammatory and homeostatic events within their infection sites. Herein we show that individual phagocytes of the jawless vertebrate Petromyzon marinus (sea lamprey), like those of teleost fish and mice, display the capacity for divergent pro-inflammatory and homeostatic responses following internalization of zymosan and apoptotic cells, respectively. Professional phagocytes (macrophages, monocytes, neutrophils) were the primary contributors to the internalization of pro-inflammatory particles among goldfish (C. auratus) and lamprey (P. marinus) hematopoietic leukocytes. However, goldfish showed a greater ability for zymosan phagocytosis when compared to their jawless counterparts. Coupled to this increase was a significantly lower sensitivity of goldfish phagocytes to homeostatic signals derived from apoptotic cell internalization. Together, this translated into a significantly greater capacity for induction of antimicrobial respiratory burst responses compared to lamprey phagocytes, but also a decreased efficacy in apoptotic cell-driven leukocyte homeostatic mechanisms that attenuate this pro-inflammatory process. Overall, our results show the long-standing evolutionary contribution of intrinsic phagocyte mechanisms for the control of inflammation, and illustrate one effective evolutionary strategy for increased responsiveness against invading pathogens. In addition, they highlight the need for development of complementary regulatory mechanisms of inflammation to ensure continued maintenance of host integrity amidst increasing challenges from invading pathogens.

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.)

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC(50)) values of 199 μmol l(-1) and 4132 μmol l(-1) for NH(3) and total ammonia ([T(Amm)]=[NH(3)]+[NH(4)(+)]), respectively. These values were ~5-6 times greater than corresponding NH(3) and T(Amm) LC(50) values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH(4)Cl (3-5 mmol l(-1)) for 5 days, despite 6-fold increases in plasma [T] to ~1300 μmol l(-1) and 3-fold increases in brain [T(Amm)] to 6700 μmol l(-1). Muscle [T(Amm)] increased by almost 8-fold from ~900 μmol kg(-1) wet mass (WM) to greater than 7000 μmol kg(-1) WM by 48 h, and stabilized. Although urea excretion rates (J(Urea)) increased by 2-3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000 μmol kg(-1) WM to 15,000 μmol kg(-1) WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8 mg kg(-1)) or ethanol (1-8 mg kg(-1)) increased trout survival time by 1.5-2.0-fold during exposure to 2 mmol l(-1) ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8-9.5 mmol l(-1) T(Amm)) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5 mmol l(-1) or 10 mmol l(-1) T(Amm) reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40-60% lower in goldfish exposed to 3-5 mmol l(-1) T(Amm) for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O(2) consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50μmolL(-1) TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The present study examined the effect of dissolved organic carbon (DOC), both added and inherent, on Cu toxicity in glochidia, the larvae of freshwater mussels. Using incremental additions of natural DOC concentrate and reconstituted water, a series of acute copper toxicity tests were conducted. An increase in DOC from 0.7 to 4.4 mg  C/L resulted in a fourfold increase (36-150 µg Cu/L) in the 24-h median effective concentration (EC50) and a significant linear relationship (r² = 0.98, p = 0.0008) between the DOC concentration and the Cu EC50 of Lampsilis siliquoidea glochidia. The ameliorating effect of added DOC on Cu toxicity was confirmed using a second mussel species, the endangered (in Canada) Lampsilis fasciola. The effect of inherent (i.e., not added) DOC on Cu toxicity was also assessed in eight natural waters (DOC 5-15 mg C/L). These experiments revealed a significant relationship between the EC50 and the concentration of inherent DOC (r² = 0.79, p = 0.0031) with EC50s ranging from 27 to 111 µg Cu/L. These laboratory tests have demonstrated that DOC provides glochidia with significant protection from acute Cu toxicity. The potential risk that Cu poses to mussel populations was assessed by comparing Cu and DOC concentrations from significant mussel habitats in Ontario to the EC50s. Although overall mean Cu concentration in the mussel's habitat was well below the acutely toxic level given the concentration of DOC, episodic Cu releases in low DOC waters may be a concern for the recovery of endangered freshwater mussels. The results are examined in the context of current Cu water quality regulations including the U.S. Environmental Protection Agency's (U.S. EPA) biotic ligand model., (© 2010 SETAC.)

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.)

Although the pesticide, 3-trifluoromethyl-4-nitrophenol (TFM), has been extensively used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes, it is surprising that its mechanism(s) of toxicity is unresolved. A better knowledge of the mode of toxicity of this pesticide is needed for predicting and improving the effectiveness of TFM treatments on lamprey, and for risk assessments regarding potential adverse effects on invertebrate and vertebrate non-target organisms. We investigated two hypotheses of TFM toxicity in larval sea lamprey. The first was that TFM interferes with oxidative ATP production by mitochondria, causing rapid depletion of energy stores in vital, metabolically active tissues such as the liver and brain. The second was that TFM toxicity resulted from disruption of gill-ion uptake, adversely affecting ion homeostasis. Exposure of larval sea lamprey to 4.6 m gl(-1) TFM (12-h LC50) caused glycogen concentrations in the brain to decrease by 80% after 12h, suggesting that the animals increased their reliance on glycolysis to generate ATP due to a shortfall in ATP supply. This conclusion was reinforced by a 9-fold increase in brain lactate concentration, a 30% decrease in brain ATP concentration, and an 80% decrease in phosphocreatine (PCr) concentration after 9 and 12h. A more pronounced trend was noted in the liver, where glycogen decreased by 85% and ATP was no longer detected after 9 and 12h. TFM led to marginal changes in whole body Na(+), Cl(-), Ca(2+) and K(+), as well as in plasma Na(+) and Cl(-), which were unlikely to have contributed to toxicity. TFM had no adverse effect on Na(+) uptake rates or gill Na(+)/K(+)-ATPase activity. We conclude that TFM toxicity in the sea lamprey is due to a mismatch between ATP consumption and ATP production rates, leading to a depletion of glycogen in the liver and brain, which ultimately leads to neural arrest and death.

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.

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Models such as the Biotic Ligand Model (BLM) predict how natural organic matter (NOM) and competing ions (e.g., Ca(2+), H(+) and Na(+)) affect metal bioavailability and toxicity in aquatic organisms. However, such models focus upon individual metals, not metal mixtures. This study determined whether Pb and Cd interact at the gill of rainbow trout (Oncorhynchus mykiss) when trout were exposed to environmentally relevant concentrations of these metals (Cd<100 nmol L(-1); Pb<500 nmol L(-1)) in soft (<100 micromol Ca(2+)L(-1)), moderately acidic (pH 6.0) water. The 96-h LC50 for Pb was 482 nmol L(-1), indicating that Pb was one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, 6.7 nmol L(-1). Surprisingly, fish acclimated to soft water had multiple populations of Pb-gill and Cd-gill binding sites. A low capacity, high affinity population of Pb-gill binding sites had a B(max) of 18.2 nmol g(-1) wet weight (ww) and apparent K(Pb-gill)=7.05, but a second low affinity population could not be saturated up to free Pb concentrations approaching 4000 nmol L(-1). Two populations of Cd-gill binding sites were characterized: a high affinity, low capacity population with an apparent K(Cd-gill)=7.33 and B(max)=1.73 nmol g(-1) ww, and a low affinity, high capacity population with an apparent K(Cd-gill)=5.86, and B(max)=13.7 nmol g(-1) ww. At low concentrations, Cd plus Pb accumulation was less than additive because Cd out-competed Pb for gill binding sites, which were likely apical Ca(2+)-channels. While disturbances to Ca(2+) influx were caused by Cd alone, Pb alone had no effect. However, Pb exacerbated Cd-induced disturbances to Ca(2+) influx demonstrating that, although Pb- plus Cd-gill binding was less than additive due to competition, the effects (ionic disturbances) were more than additive (synergistic). Pb was also likely binding to intracellular targets, such as branchial carbonic anhydrase, which led to inhibited Na(+) influx. This ionic disturbance was exacerbated by Cd. We conclude that exposure to environmentally relevant concentrations of Pb plus Cd results in less than additive metal-gill binding in soft, moderately acidic waters. However, ionic disturbances caused by Cd plus Pb are greater than additive, and this may ultimately increase the toxicity of Cd-Pb mixtures to fishes. Our findings suggest that it may be necessary to re-evaluate water quality criteria and assumptions of the BLM for fish exposed to mixtures of Pb and Cd in the acidic, soft waters found in the Canadian Shield, Scandinavia and other sensitive regions.

Although the number of fish species that have been studied for both hypoxia/anoxia tolerance and ammonia tolerance are few, there appears to be a correlation between the ability to survive these two insults. After establishing this correlation with examples from the literature, and after examining the role Peter Lutz played in catalyzing this convergent interest in two variables, this article explores potential mechanisms underpinning this correlation. We draw especially on the larger body of information for two human diseases with the same effected organ (brain), namely stroke and hepatic encephalopathy. While several dissimilarities exist between the responses of vertebrates to anoxia and hyperammonemia, one consistent observation in both conditions is an overactivation of NMDA receptors or glutamate neurotoxicity. We propose a glutamate excitotoxicity hypothesis to explain the correlation between ammonia and hypoxia resistance in fish. Furthermore, we suggest several experimental paths to test this hypothesis.

Although urea production and metabolism in lungfish have been thoroughly studied, we have little knowledge of how internal osmotic and electrolyte balance are controlled during estivation or in water. We tested the hypothesis that, compared with the body surface of teleosts, the slender African lungfish (Protopterus dolloi) body surface was relatively impermeable to water, Na(+), and Cl(-) due to its greatly reduced gills. Accordingly, we measured the tritiated water ((3)H-H(2)O) flux in P. dolloi in water and during air exposure. In water, (3)H-H(2)O efflux was comparable with the lowest measurements reported in freshwater teleosts, with a rate constant (K) of 17.6% body water h(-1). Unidirectional ion fluxes, measured using (22)Na(+) and (36)Cl(-), indicated that Na(+) and Cl(-) influx was more than 90% lower than values reported in most freshwater teleosts. During air exposure, a cocoon formed within 1 wk that completely covered the dorsolateral body surface. However, there were no disturbances to blood osmotic or ion (Na(+), Cl(-)) balance, despite seven- to eightfold increases in plasma urea after 20 wk. Up to 13-fold increases in muscle urea (on a dry-weight basis) were the likely explanation for the 56% increase in muscle water content observed after 20 wk of air exposure. The possibility that muscle acted as a "water reservoir" during air exposure was supported by the 20% decline in body mass observed during subsequent reimmersion in water. This decline in body mass was equivalent to 28 mL water in a 100-g animal and was very close to the calculated net water gain (approximately 32 mL) observed during the 20-wk period of air exposure. Tritiated water and unidirectional ion fluxes on air-exposed lungfish revealed that the majority of water and ion exchange was via the ventral body surface at rates that were initially similar to aquatic rates. The (3)H-H(2)O flux declined over time but increased upon reimmersion. We conclude that the slender lungfish body surface, including the gills, has relatively low permeability to water and ions but that the ventral surface is an important site of osmoregulation and ionoregulation. We further propose that an amphibian-like combination of ventral skin water and ion permeability, plus internal urea accumulation during air exposure, allows P. dolloi to extract water from its surroundings and to store water in the muscle when the water supply becomes limited.

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey's life cycle.

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.

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.)

Although the majority of the sea lamprey's (Petromyzon marinus) life cycle is spent as a burrow-dwelling larva, or ammocoete, surprisingly little is known about intermediary metabolism in this stage of the lamprey's life history. In this study, larval sea lampreys (ammocoetes) were vigorously exercised for 5 min, and their patterns of metabolic fuel depletion and replenishment and oxygen consumption, along with measurements of net whole-body acid and ion movements, were followed during a 4-24-h postexercise recovery period. Exercise led to initial five- to sixfold increases in postexercise oxygen consumption, which remained significantly elevated by 1.5-2.0 times for the next 3 h. Exercise also led to initial 55% drops in whole-body phosphocreatine, which was restored by 0.5 h, but no significant changes in whole-body adenosine triphosphate were observed. Whole-body glycogen concentrations dropped by 70% immediately following exercise and were accompanied by a simultaneous ninefold increase in lactate. Glycogen and lactate were quickly restored to resting levels after 0.5 and 2.0 h, respectively. The presence of an associated metabolic acidosis was supported by very high rates of metabolic acid excretion, which approached 1,000 nmol g(-1) during the first 2 h of postexercise recovery. Exercise-induced ion imbalances were also rapidly alleviated, as initially high rates of net Na(+) and Cl(-) loss (-1,200 nmol g(-1) h(-1) and -1,800 nmol g(-1) h(-1), respectively) were corrected within 1-2 h. Although larval sea lampreys spend most of their time burrowed, they are adept at performing and recovering from vigorous anaerobic exercise. Such attributes could be important when these animals are vigorously swimming or burrowing as they evade predators or forage.

To test the hypothesis that internal ion imbalances at high pH are caused by altered branchial ion transporting capacity and permeability, radiotracers (24Na+ and 36Cl-) were used to measure ion movements across the gills of intact rainbow trout (Oncorhynchus mykiss) during 3 d exposure to pH 9.5. At control pH (pH 8.0), the trout were in net ion balance, but by 8 h at high pH, 60%-70% reductions in Cl- influx (JClin) and Na+ influx (JNain) led to net Cl- and Na+ losses of -200 micromol kg-1 h-1. Outflux (diffusive efflux plus renal ion losses) was not initially altered. By 72 h, net Cl- balance was reestablished because of a restoration of JClin. Although JNain remained 50% lower at this time, counterbalancing reductions in Na+ outflux restored net Na+ balance. One-substrate ion-uptake kinetics analyses indicated that reduced ion influx after 8 h at pH 9.5 was caused by 50% decreases in Cl- and Na+ maximal transport rates (JClmax, JNamax), likely reflecting decreased numbers of functional transport sites. Two-substrate kinetic analyses indicated that reduced internal HCO-3 and H+ supply for respective branchial Cl-/base and Na+/acid transport systems also contributed to lower JClin and, to a lesser extent, lower JNain at pH 9.5. Recovery of JClmax after 3 d accounted for restoration of Cl- balance and likely reflected increased numbers of transport sites. In contrast, JNamax remained 33% lower after 3 d, but a lower affinity of the gills for Na+ (fourfold greater KNam) accounted for the chronic reduction in Na+ influx at pH 9.5. Thus, reestablishment of Cl- uptake capacity and counterbalancing reductions in Na+ outflux allows rainbow trout to reestablish net ion balance in alkaline waters.