Upstream migrant sea lamprey (Petromyzon marinus) show signs of increasing oxidative stress but maintain aerobic capacity with age.

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.

Niclosamide Is a Much More Potent Toxicant of Mitochondrial Respiration than TFM in the Invasive Sea Lamprey (Petromyzon marinus).

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 EC50 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.

Disturbances to energy metabolism in juvenile lake sturgeon (Acipenser fulvescens) following exposure to niclosamide.

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 LC50 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.

Potential changes to the biology and challenges to the management of invasive sea lamprey Petromyzon marinus in the Laurentian Great Lakes due to climate change.

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.

Assessing off-target cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol using novel lake sturgeon cell lines.

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 ß-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 EC50 values were compared between the cell lines and to the reported in vivo LC50s. Tissues sensitive to the effects of TFM in vivo correlated with cell lines from the same tissues being most sensitive to TFM in vitro. EC50 values for the LSliver-e cells was significantly lower than the EC50 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.

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

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

Relationship between oxidative stress and brain swelling in goldfish (Carassius auratus) exposed to high environmental ammonia.

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 NH4Cl) 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.

Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition.

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 (Vm) 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 (APf) of the stellate neurons increased 68-fold. Furthermore, the GABAA 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 EGABA was below the action potential threshold (-36 mV pyramidal and -38 mV stellate neurons). Inhibition of GABAA receptors with gabazine reversed the anoxia-mediated response. While GABAB receptor inhibition alone did not affect the anoxic response, co-antagonism of GABAA and GABAB receptors (gabazine and CGP-55848) led to the generation of seizure-like activities in both neuron types. We conclude that with anoxia, Vm depolarizes towards EGABA which increases APf in stellate neurons and decreases APf in pyramidal neurons, and that GABA plays an important role in the anoxia tolerance of goldfish brain.

Adaptations of a deep sea scavenger: high ammonia tolerance and active NH4⁺ excretion by the Pacific hagfish (Eptatretus stoutii).

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.

Reversible brain swelling in crucian carp (Carassius carassius) and goldfish (Carassius auratus) in response to high external ammonia and anoxia.

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.

Interactions of Pb and Cd mixtures in the presence or absence of natural organic matter with the fish gill.

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.

Reversible disruptions to energy supply and acid-base balance in larval sea lamprey exposed to the pesticide: Niclosamide (2',5-dichloro-4'-nitrosalicylanilide).

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 LC50 (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.

Dining on the dead in the deep: Active NH4 + excretion via Na+ /H+ (NH4 + ) exchange in the highly ammonia tolerant Pacific hagfish, Eptatretus stoutii.

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 NH4 + 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 NH4 + against steep inwardly directed ENH4 + (ΔENH4 + ~ 35 mV) and pNH3 (ΔpNH3 ~ 2000 µtorr) gradients. Active NH4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na+ , indicating a Na+ /NH4 + exchange mechanism. Active NH4 + 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.

Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN 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 (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm 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 Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea 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.

The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish.

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.

A Toxic Unit and Additive Index Approach to Understanding the Interactions of 2 Piscicides, 3-Trifluoromethyl-4-Nitrophenol and Niclosamide, in Rainbow Trout.

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.

The lampricide 3-trifluoromethyl-4-nitrophenol causes temporary metabolic disturbances in juvenile lake sturgeon (Acipenser fulvescens): implications for sea lamprey control and fish conservation.

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 LC99.9), 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.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

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 (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea 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 of invasive sea lampreys using the piscicides TFM and niclosamide: Toxicology, successes & future prospects.

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.

Mitigation of lampricide toxicity to juvenile lake sturgeon: the importance of water alkalinity and life stage.

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 (14C-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 CaCO3) to moderate alkalinity (~150 mg L-1 as CaCO3), before plateauing at high alkalinity (~250 mg L-1 as CaCO3). Toxicity curves revealed that the 12-h LC50 and 12-h LC99.9 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 = LC99.9) 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.