Heatwave resilience of juvenile white sturgeon is associated with epigenetic and transcriptional alterations.

Heatwaves are increasing in frequency and severity, posing a significant threat to organisms globally. In aquatic environments heatwaves are often associated with low environmental oxygen, which is a deadly combination for fish. However, surprisingly little is known about the capacity of fishes to withstand these interacting stressors. This issue is particularly critical for species of extreme conservation concern such as sturgeon. We assessed the tolerance of juvenile white sturgeon from an endangered population to heatwave exposure and investigated how this exposure affects tolerance to additional acute stressors. We measured whole-animal thermal and hypoxic performance and underlying epigenetic and transcriptional mechanisms. Sturgeon exposed to a simulated heatwave had increased thermal tolerance and exhibited complete compensation for the effects of acute hypoxia. These changes were associated with an increase in mRNA levels involved in thermal and hypoxic stress (hsp90a, hsp90b, hsp70 and hif1a) following these stressors. Global DNA methylation was sensitive to heatwave exposure and rapidly responded to acute thermal and hypoxia stress over the course of an hour. These data demonstrate that juvenile white sturgeon exhibit substantial resilience to heatwaves, associated with improved cross-tolerance to additional acute stressors and involving rapid responses in both epigenetic and transcriptional mechanisms.

Identification of upper thermal thresholds during development in the endangered Nechako white sturgeon with management implications for a regulated river.

Climate change-induced warming effects are already evident in river ecosystems, and projected increases in temperature will continue to amplify stress on fish communities. In addition, many rivers globally are impacted by dams, which have many negative effects on fishes by altering flow, blocking fish passage, and changing sediment composition. However, in some systems, dams present an opportunity to manage river temperature through regulated releases of cooler water. For example, there is a government mandate for Kenney dam operators in the Nechako river, British Columbia, Canada, to maintain river temperature <20°C in July and August to protect migrating sockeye salmon (Oncorhynchus nerka). However, there is another endangered fish species inhabiting the same river, Nechako white sturgeon (Acipenser transmontanus), and it is unclear if these current temperature regulations, or timing of the regulations, are suitable for spawning and developing sturgeon. In this study, we aimed to identify upper thermal thresholds in white sturgeon embryos and larvae to investigate if exposure to current river temperatures are playing a role in recruitment failure. We incubated embryos and yolk-sac larvae in three environmentally relevant temperatures (14, 18 and 21°C) throughout development to identify thermal thresholds across different levels of biological organization. Our results demonstrate upper thermal thresholds at 21°C across physiological measurements in embryo and yolk-sac larvae white sturgeon. Before hatch, both embryo survival and metabolic rate were reduced at 21°C. After hatch, sublethal consequences continued at 21°C because larval sturgeon had decreased thermal plasticity and a dampened transcriptional response during development. In recent years, the Nechako river has reached 21°C by the end of June, and at this temperature, a decrease in sturgeon performance is evident in most of the traits measured. As such, the thermal thresholds identified here suggest current temperature regulations may not be suitable for developing white sturgeon and future recruitment.

The role of salinity in recovery of white sturgeon (Acipenser transmontanus) from stimulated angling stress.

White sturgeon (Acipenser transmontanus) in the Lower Fraser River are the focus of a catch-and-release angling fishery in British Columbia, Canada. However, the lower region of the catch area includes areas where tidal waters invade, and the consequence of salinity levels on recovery from an angling challenge are not characterized in sturgeon, despite theoretical implications of its import. We acclimated white sturgeon to various salinities (0, 10 and 20‰ (parts per thousand)) to investigate the effects of acclimation on recovery from stimulated angling stress that was induced through manual chasing. This challenge elicited the traditional physiological responses such as ion homeostasis disturbance, increases in secondary stress indicators and metabolic acidosis; however, environmental salinity altered the timing of recovery in some of the parameters measured. In addition, the severity of the intracellular pH disturbance in both heart and red blood cell seemed to be mediated in fresh water, yet the recovery pattern of plasma chloride and bicarbonate ions seemed to be facilitated by higher salinity. In general, responses were similar but not identical, leading us to conclude that the role of salinity on recovery from exercise is complex but not insignificant. Salinity may be important to behaviours exhibited by white sturgeon (such as migrations) in their respective saline environments, but less so around the impact of an angling stressor. Further exploration of this response may provide insight on whether the current tidal boundaries for angling white sturgeon are appropriate.

Designing flows to enhance ecosystem functioning in heavily altered rivers.

More than a century of dam construction and water development in the western United States has led to extensive ecological alteration of rivers. Growing interest in improving river function is compelling practitioners to consider ecological restoration when managing dams and water extraction. We developed an Ecological Response Model (ERM) for the Cache la Poudre River, northern Colorado, USA, to illuminate effects of current and possible future water management and climate change. We used empirical data and modeled interactions among multiple ecosystem components to capture system-wide insights not possible with the unintegrated models commonly used in environmental assessments. The ERM results showed additional flow regime modification would further alter the structure and function of Poudre River aquatic and riparian ecosystems due to multiple and interacting stressors. Model predictions illustrated that specific peak flow magnitudes in spring and early summer are critical for substrate mobilization, dynamic channel morphology, and overbank flows, with strong subsequent effects on instream and riparian biota that varied seasonally and spatially, allowing exploration of nuanced management scenarios. Instream biological indicators benefitted from higher and more stable base flows and high peak flows, but stable base flows with low peak flows were only half as effective to increase indicators. Improving base flows while reducing peak flows, as currently proposed for the Cache la Poudre River, would further reduce ecosystem function. Modeling showed that even presently depleted annual flow volumes can achieve substantially different ecological outcomes in designed flow scenarios, while still supporting social demands. Model predictions demonstrated that implementing designed flows in a natural pattern, with attention to base and peak flows, may be needed to preserve or improve ecosystem function of the Poudre River. Improved regulatory policies would include preservation of ecosystem-level, flow-related processes and adaptive management when water development projects are considered.

Repeated measurement of Mo2 in small aquatic organisms: a manual intermittent flow respirometer using off-the-shelf components.

Measurement of rates of oxygen consumption ( Mo2) in small aquatic embryos or larvae (<1 mm) in response to altered environmental conditions has traditionally been challenging. Here, using modifications of a commercially available fluorescent optode flow-through cell (FTC; PreSens FTC-PSt3) and routine laboratory supplies (syringes, stopcocks, tubing), we have constructed a manual intermittent flow respirometer (MIFR) that allows measurement of Mo2 in small numbers of individuals when sequentially exposed to different environmental conditions (e.g., changes in seawater pH) through a gravity-driven media replacement perfusion system. We first show that the FTC can be used in "static" mode while incubating small numbers of embryos/larvae contained within the planar oxygen sensor (POS) chamber with Nitex filters. We then demonstrate the use of the MIFR by exposing larval echinoderms ( Fellaster zelandiae, Evechinus chloroticus, and Centrostephanus rodgersii) to seawater equilibrated with elevated CO2 and measured Mo2 during acute and chronic exposure to hypercapnia. This MIFR method will allow investigators to address questions regarding the respiratory physiology of small aquatic animals, such as the thresholds for metabolic depression in embryonic and larval forms. NEW & NOTEWORTHY A manual intermittent flow respirometer (MIFR), allowing media exchange in a flow-through cell containing small aquatic organisms, permits repeated measurement of Mo2 of individuals not only in a single medium (e.g., technical replication), but also in different media (here, high CO2-equilibrated seawater), enabling measurement of acute physiological responses to changed conditions. This versatile technique has wide-ranging implications for the study of the Mo2 response of aquatic organisms in the face of climate change.

Preferential intracellular pH regulation: hypotheses and perspectives.

The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land.

Hagfish: Champions of CO2 tolerance question the origins of vertebrate gill function.

The gill is widely accepted to have played a key role in the adaptive radiation of early vertebrates by supplanting the skin as the dominant site of gas exchange. However, in the most basal extant craniates, the hagfishes, gills play only a minor role in gas exchange. In contrast, we found hagfish gills to be associated with a tremendous capacity for acid-base regulation. Indeed, Pacific hagfish exposed acutely to severe sustained hypercarbia tolerated among the most severe blood acidoses ever reported (1.2 pH unit reduction) and subsequently exhibited the greatest degree of acid-base compensation ever observed in an aquatic chordate. This was accomplished through an unprecedented increase in plasma [HCO3(-)] (>75 mM) in exchange for [Cl(-)]. We thus propose that the first physiological function of the ancestral gill was acid-base regulation, and that the gill was later co-opted for its central role in gas exchange in more derived aquatic vertebrates.

Could thermal sensitivity of mitochondria determine species distribution in a changing climate?

For many aquatic species, the upper thermal limit (Tmax) and the heart failure temperature (THF) are only a few degrees away from the species' current environmental temperatures. While the mechanisms mediating temperature-induced heart failure (HF) remain unresolved, energy flow and/or oxygen supply disruptions to cardiac mitochondria may be impacted by heat stress. Recent work using a New Zealand wrasse (Notolabrus celidotus) found that ATP synthesis capacity of cardiac mitochondria collapses prior to T(HF). However, whether this effect is limited to one species from one thermal habitat remains unknown. The present study confirmed that cardiac mitochondrial dysfunction contributes to heat stress-induced HF in two additional wrasses that occupy cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats. With exposure to heat stress, T. lunare had the least scope to maintain heart function with increasing temperature. Heat-exposed fish of all species showed elevated plasma succinate, and the heart mitochondria from the cold temperate N. fucicola showed decreased phosphorylation efficiencies (depressed respiratory control ratio, RCR), cytochrome c oxidase (CCO) flux and electron transport system (ETS) flux. In situ assays conducted across a range of temperatures using naive tissues showed depressed complex II (CII) and CCO capacity, limited ETS reserve capacities and lowered efficiencies of pyruvate uptake in T. lunare and N. celidotus. Notably, alterations of mitochondrial function were detectable at saturating oxygen levels, indicating that cardiac mitochondrial insufficiency can occur prior to HF without oxygen limitation. Our data support the view that species distribution may be related to the thermal limits of mitochondrial stability and function, which will be important as oceans continue to warm.

Low-O2 acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function.

It was hypothesised that chronic hypoxia acclimation (preconditioning) would alter the behavioural low-O(2) avoidance strategy of fish as a result of both aerobic and anaerobic physiological adaptations. Avoidance and physiological responses of juvenile snapper (Pagrus auratus) were therefore investigated following a 6 week period of moderate hypoxia exposure (10.2-12.1 kPa P(O(2)), 21 ± 1 °C) and compared with those of normoxic controls (P(O(2))=20-21 kPa, 21 ± 1 °C). The critical oxygen pressure (P(crit)) limit of both groups was unchanged at ~7 kPa, as were standard, routine and maximum metabolic rates. However, hypoxia-acclimated fish showed increased tolerances to hypoxia in behavioural choice chambers by avoiding lower P(O(2)) levels (3.3 ± 0.7 vs 5.3 ± 1.1 kPa) without displaying greater perturbations of lactate or glucose. This behavioural change was associated with unexpected physiological adjustments. For example, a decrease in blood O(2) carrying capacity was observed after hypoxia acclimation. Also unexpected was an increase in whole-blood P(50) following acclimation to low O(2), perhaps facilitating Hb-O(2) off-loading to tissues. In addition, cardiac mitochondria measured in situ using permeabilised fibres showed improved O(2) uptake efficiencies. The proportion of the anaerobic enzyme lactate dehydrogenase, at least relative to the aerobic marker enzyme citrate synthase, also increased in heart and skeletal red muscle, indicating enhanced anaerobic potential, or in situ lactate metabolism, in these tissues. Overall, these data suggest that a prioritization of O(2) delivery and O(2) utilisation over O(2) uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O(2) tolerance.

Metabolic changes associated with acid-base regulation during hypercarbia in the CO2-tolerant chondrostean, white sturgeon (Acipenser transmontanus).

CO(2) tolerance in white sturgeon is associated with the ability to tightly regulate intracellular pH (pHi) despite a large reduction in extracellular pH (pHe) termed preferential pHi regulation. How this regulatory response affects whole animal metabolic rate is unknown. Accordingly, we characterized oxygen consumption rate ( [Formula: see text] ) and metabolically-relevant organismal and cellular responses in white sturgeon during exposure to hypercarbia. White sturgeon were able to protect intracellular pH (pHi) in liver and white muscle as early as 6h (the earliest time period investigated) following exposure to severe (sub-lethal) hypercarbia (45 and 90 mm Hg PCO(2)). Sturgeon exposed to 15 and 30 mm Hg PCO(2) exhibited pHe compensation and significant increases in [Formula: see text] (up to 80% greater than control values). In contrast, severe hypercarbia (≥45 mm Hg PCO(2)) elicited an uncompensated reduction in pHe (up to ~1.0 pH units) and red blood cells (as great as ~0.5 pH units), and was accompanied by 30 and 60% reductions in [Formula: see text] , respectively. While behavioral, respiratory and cellular responses to hypercarbia were observed, none corresponded well with the pattern or magnitude of changes in [Formula: see text] . The findings of this research provide empirical support for the hypothesis that preferential pHi regulation is not metabolically costly, and thus may have been a strategy strongly selected for in fishes encountering short-term hypercarbia.

Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line, RTH 149.

Fish exposed to elevated water CO(2) experience a rapid increase in blood CO(2) levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pH(i)) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pH(i) regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pH(i) regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO(2) or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO(2) was maintained. In addition, rates of pH(i) recovery from NH(4)Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO(2) indirectly impeded pH(i) recovery through a reduction in pH(e) and/or pH(i). Moreover, pH(i) regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pH(i) regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid-base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pH(i) recovery rates relative to primary hepatocytes.

The effects of temperature on the physiological response to low oxygen in Atlantic sturgeon.

Atlantic sturgeon (Acipenser oxyrhynchus), which are bottom dwelling and migratory fish, experience environmental hypoxia in their natural environment. Atlantic sturgeon, acclimated to either 5 or 15°C, were subjected to a 1 h severe (<10 mm Hg) hypoxia challenge in order to document their physiological responses. We measured hematological parameters, including O(2) transport (hemoglobin, hematocrit), ionic (chloride, osmolality), and metabolic (glucose, lactate) variables under normoxic conditions (~160 mm Hg), immediately following a 1 h exposure to hypoxic water, and following a further 2 h of recovery from this challenge in normoxic water. In a second experiment, we assessed the opercular beat frequency before, during, and after hypoxic exposure. Hemoglobin concentrations and hematocrit were significantly different between fish held at 5°C vs. 15°C and also significantly different between normoxia prior to hypoxia and following recovery. Plasma lactate concentrations increased following hypoxia at both temperatures, indicative of an increase in anaerobic metabolism. In contrast, a significant increase in plasma glucose concentrations in response to hypoxia only occurred at 5°C, suggesting different fuel demands under different temperatures. Changes in opercular beat frequency (OBF) were dependent on temperature. At 5°C, OBF increased upon exposure to hypoxia, but returned to pre-exposure levels within 35 min for the remainder of the experiment. During hypoxia at 15°C, OBF increased very briefly, but then rapidly (within 20 min) decreased to levels below control values. Following a return to normoxia, OBF quickly increased to control levels. Overall, these findings suggest that Atlantic sturgeons are relatively tolerant to short-term and severe hypoxic stress, and the strategies for hypoxia tolerance may be temperature dependent.

Exceptional CO2 tolerance in white sturgeon (Acipenser transmontanus) is associated with protection of maximum cardiac performance during hypercapnia in situ.

White sturgeon rank among the most CO2-tolerant fish species examined to date. We investigated whether this exceptional CO2 tolerance extended to the heart, an organ generally viewed as acidosis intolerant. Maximum cardiac output (Q(max)) and maximum cardiac power output (PO(max)) were assessed using a working, perfused, in situ heart preparation. Exposure to a Pco2 of 3 kPa for 20 min had no significant effect on maximum cardiac performance, while exposure to 6-kPa Pco2 reduced heart rate, Q(max), PO(max), and rate of ventricular force generation (F(O)) by 23%, 28%, 26%, and 18%, respectively; however, full recovery was observed in all these parameters upon return to control conditions. These modest impairments during exposure to 6-kPa Pco2 were associated with partially compensated intracellular ventricular acidosis. Maximum adrenergic stimulation (500 nmol L⁻¹ adrenaline) during 6-kPa Pco2 protected maximum cardiac performance via increased inotropy (force of contraction) without affecting heart rate. Exposure to higher CO2 levels associated with morbidity in vivo (i.e., 8-kPa Pco2) induced arrhythmia and a reduction in stroke volume during power assessment. Clearly, white sturgeon hearts are able to increase cardiac performance during severe hypercapnia that is lethal to other fishes. Future work focusing on atypical aspects of sturgeon cardiac function, including the lack of chronotropic response to adrenergic stimulation during hypercapnia, is warranted.

Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells.

Fish, exposed to elevated water CO(2), experience a rapid elevation in blood CO(2) (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. White sturgeon, Acipenser transmontanus, are exceptionally CO(2) tolerant and can regulate tissue intracellular pH (pH(i)) in the presence of a pronounced hypercapnic acidosis (preferential pH(i) regulation). In this study, pH(i) regulatory capacity of sturgeon liver cells in primary culture was examined to assess the suitability of employing this in vitro system to understand in vivo CO(2) tolerance in sturgeon. Using the pH-sensitive fluoroprobe BCECF, real-time changes in resting pH(i) and rates of pH(i) recovery were investigated during exposure to hypercapnia (3 and 6% CO(2)) in the absence and presence of additional acid loads induced by (20 mM) ammonium prepulse. During short-term (10 min) exposure to hypercapnia (3 and 6% CO(2)), sturgeon cells were acidified and no pH(i) compensation was observed. However, when exposure to 6% CO(2) was extended to over 19 h, the CO(2)-induced intracellular acidosis was partially compensated by a pH(i) increase of over 0.2 pH unit despite the sustained extracellular acidosis, indicative of a capacity for preferential pH(i) regulation in vitro. Since this capacity in sturgeon liver is present both in vivo and in vitro, the transmembrane transporters involved may be the same. Therefore, cell culture may be a suitable tool to identify the transporters (i.e., the cellular mechanisms underlying in vivo CO(2) tolerance) in white sturgeon and possibly in other hypercapnia-tolerant species.

Intrinsic mechanical properties of the perfused armoured catfish heart with special reference to the effects of hypercapnic acidosis on maximum cardiac performance.

The armoured catfish, Pterygoplichthys pardalis, is known to be extremely tolerant of environmental hypercarbia (elevated water CO(2) tensions), which occurs in their natural environment. In addition, previous studies have demonstrated that during exposure to hypercarbia, P. pardalis does not exhibit extracellular pH compensation and thus the heart and other organs must continue to function despite a severe extracellular acidosis. We used an in situ perfused heart preparation to determine the effects of an extracellular hypercapnic (elevated CO(2) in the animal) acidosis (1-7.5% CO(2)) on heart function, specifically cardiac output, power output, heart rate and stroke volume. The present study is the first to comprehensively examine cardiac function in an acidosis-tolerant teleost. When compared with control conditions, maximum cardiac performance was unaffected at levels of CO(2) as high as 5%, far exceeding the hypercapnic tolerance of other teleosts. Moreover, P. pardalis exhibited only a moderate decrease (approximately 35%) in cardiac performance when exposed to 7.5% CO(2), and full cardiac performance was restored in six out of seven hearts upon return to control conditions. Myocardial intracellular pH (pH(i)) was protected in situ, as has been found in vivo, and this protection extended to the highest level of CO(2) (7.5%) investigated. Thus, maintained heart function during a hypercapnic acidosis in P. pardalis is probably associated with preferential pH(i) regulation of the heart, but ultimately is not sufficient to prevent loss of cardiac function. Our findings suggest the need for further study to elucidate the mechanisms behind this remarkable cardiac hypercapnic tolerance.

Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer.

We have explored the molecular and physiological responses of the euryhaline killifish Fundulus heteroclitus to transfer from brackish water (10% seawater) to 100% seawater for 12 h, 3 days or 7 days. Plasma [Na+] and [Cl-] were unchanged after transfer, and plasma cortisol underwent a transient increase. Na+/K+-ATPase activity increased 1.5-fold in the gills and opercular epithelium at 7 days (significant in gills only), responses that were preceded by three- to fourfold increases in Na+/K+-ATPase alpha(1a) mRNA expression. Expression of Na+/K+/2Cl- cotransporter 1, cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, Na+/H+-exchanger 3 (significant in opercular epithelium only) and carbonic anhydrase II mRNA also increased two- to fourfold after transfer. Drinking rate increased over twofold after 12 h and remained elevated for at least 7 days. Surprisingly, net rates of water and ion absorption measured in vitro across isolated intestines decreased approximately 50%, possibly due to reduced salt demands from the diet in seawater, but water absorption capacity still exceeded the drinking rate. Changes in bulk water absorption were well correlated with net ion absorption, and indicated that slightly hyperosmotic solutions (>or=298 mmol l(-1)) were transported. There were no reductions in unidirectional influx of Na+ from luminal to serosal fluid or intestinal Na+/K+-ATPase activity after transfer. Overall, our results indicate that gill and opercular epithelia function similarly at a molecular level in seawater, in contrast to their divergent function in freshwater, and reveal unexpected changes in intestinal function. As such they provide further insight into the mechanisms of euryhalinity in killifish.

An in vitro approach for modelling branchial copper binding in rainbow trout.

The main objective of this study was to characterize the individual effects of water chemistry (Ca(2+), Na(+), dissolved organic matter (DOM), pH, alkalinity) on the rapid binding of copper to the gill surface of rainbow trout using an in vitro gill binding assay. In this assay, individual gill arches were exposed for 5 min to (64)Cu labelled copper solutions ranging from 0.02 to 0.16 microM in water chemistries reflecting the full range of fresh water values for the Great Lakes. The gills displayed saturable Cu binding within this Cu range but gill-Cu binding was completely unaffected over the full range of calcium, sodium and alkalinity concentrations used. Only low pH (pH 4.0) and commercial DOM (Aldrich humic acid at > or =3 mgC/l) altered copper binding to rainbow trout gills in vitro. These findings were consistent with the results of geochemical modelling of our water chemistry (using MINEQL+, Version 4.5) which showed that H(+) and DOM affected the free cupric ion concentration. However, DOM (up to 80 mgC/l) was only able to reduce Cu on the gills by 50%. We hypothesize that in the range of 0.02-0.16 microM Cu there are two high affinity Cu binding sites on the gills, one having a substantially higher affinity for copper than DOM. The absence of a calcium effect on gill copper binding was in accord with in vivo evidence that calcium primarily acts to alter the physiology of the gill binding sites through acclimatory processes, rather than through competitive interactions. It was a surprise that water chemistry parameters influence rapid gill-metal binding in a manner different to their influence on acute toxicity and different from the effects on long-term binding reported in other studies. Currently, the biotic ligand model uses the rapid increase of gill copper (believed to reflect binding to the physiologically active receptor sites) to model gill binding characteristics. The distinction between rapid surface binding and metal uptake obviously plays an important role in determining the toxic effects of copper, especially when regulators need to predict the modifying effects of water chemistry.

Social interactions affect physiological consequences of sublethal copper exposure in rainbow trout, Oncorhynchus mykiss.

The interactions of sublethal waterborne copper exposure and social dominance behavior were examined in juvenile rainbow trout. Dominance hierarchies were determined between pairs of fish by behavioral observations and among groups of 10 fish by the use of passive integrated transponder (PIT) tagging equipment. The present study is one of the first to utilize this novel PIT tag method for behavioral assessment. Feeding behavior was quantified by placing a PIT tag recorder at the entrance to the feeding area. Linear dominance hierarchies were documented based on these observations of feeding behavior. Dominance hierarchies established in control water were not altered by exposure to 30 microg/L of copper; however, physiological responses of fish to sublethal concentrations of copper were related to social rank. Subordinate fish exhibited a higher accumulation of copper in both gill and liver tissue. Subordinates of paired fish were also shown to have a higher uptake of sodium than dominant fish, and the uptake of sodium was correlated with uptake of copper from the water. Therefore, within a population of fish, it cannot be assumed that individuals of different social status will exhibit the same physiological responses to the presence of copper.