Metabolic rate and critical thermal maximum CTmax estimates for westslope cutthroat trout, Oncorhynchus clarkii lewisi.

Global warming is changing the thermal habitat of cold-water freshwater fishes, which can lead to decreased fitness and survival and cause shifts in species distributions. The Alberta population of westslope cutthroat trout (Oncorhynchus clarkii lewisi) is listed as 'Threatened' under the Canadian Species at Risk Act. The major threats to the species are the alteration in habitat and water flow, competition and hybridization with non-native trout species and climate change. Here, we conducted (i) intermittent-flow respirometry experiments with adult native westslope cutthroat trout and non-native rainbow trout (Oncorhynchus mykiss) and (ii) critical thermal maximum experiments (CTmax ) with adult westslope cutthroat trout to obtain valuable input data for species distribution models. For both species, standard metabolic rate (SMR) was lower at 10°C compared to 15°C and westslope cutthroat trout had higher SMR than rainbow trout. Although there were inter-specific differences in SMR, forced aerobic scope (using a standardized chase protocol) was different at 10°C, but no significant differences were observed at 15°C because of relative smaller differences in maximum metabolic rate between the species. CTmax of westslope cutthroat trout acclimated to 10°C was 27.0 ± 0.8°C and agitation temperature was 25.2 ± 1.0°C. The results from this study will inform and parametrize cumulative effects assessments and bioenergetics habitat modelling for the recovery planning of the species.

A chromosomal inversion may facilitate adaptation despite periodic gene flow in a freshwater fish.

Differences in genomic architecture between populations, such as chromosomal inversions, may play an important role in facilitating adaptation despite opportunities for gene flow. One system where chromosomal inversions may be important for eco-evolutionary dynamics is in freshwater fishes, which often live in heterogenous environments characterized by varying levels of connectivity and varying opportunities for gene flow. In the present study, reduced representation sequencing was used to study possible adaptation in n = 345 walleye (Sander vitreus) from three North American waterbodies: Cedar Bluff Reservoir (Kansas, USA), Lake Manitoba (Manitoba, Canada), and Lake Winnipeg (Manitoba, Canada). Haplotype and outlier-based tests revealed a putative chromosomal inversion that contained three expressed genes and was nearly fixed in walleye assigned to Lake Winnipeg. These patterns exist despite the potential for high gene flow between these proximate Canadian lakes, suggesting that the inversion may be important for facilitating adaptive divergence between the two lakes despite gene flow. However, a specific adaptive role for the putative inversion could not be tested with the present data. Our study illuminates the importance of genomic architecture consistent with local adaptation in freshwater fishes. Furthermore, our results provide additional evidence that inversions may facilitate local adaptation in many organisms that inhabit connected but heterogenous environments.

Molecular and physiological responses predict acclimation limits in juvenile brook trout (Salvelinus fontinalis).

Understanding the resilience of ectotherms to high temperatures is essential because of the influence of climate change on aquatic ecosystems. The ability of species to acclimate to high temperatures may determine whether populations can persist in their native ranges. We examined physiological and molecular responses of juvenile brook trout (Salvelinus fontinalis) to six acclimation temperatures (5, 10, 15, 20, 23 and 25°C) that span the thermal distribution of the species to predict acclimation limits. Brook trout exhibited an upregulation of stress-related mRNA transcripts (heat shock protein 90-beta, heat shock cognate 71 kDa protein, glutathione peroxidase 1) and downregulation of transcription factors and osmoregulation-related transcripts (nuclear protein 1, Na+/K+/2Cl- co-transporter-1-a) at temperatures ≥20°C. We then examined the effects of acclimation temperature on metabolic rate (MR) and physiological parameters in fish exposed to an acute exhaustive exercise and air exposure stress. Fish acclimated to temperatures ≥20°C exhibited elevated plasma cortisol and glucose, and muscle lactate after exposure to the acute stress. Fish exhibited longer MR recovery times at 15 and 20°C compared with the 5 and 10°C groups; however, cortisol levels remained elevated at temperatures ≥20°C after 24 h. Oxygen consumption in fish acclimated to 23°C recovered quickest after exposure to acute stress. Standard MR was highest and factorial aerobic scope was lowest for fish held at temperatures ≥20°C. Our findings demonstrate how molecular and physiological responses predict acclimation limits in a freshwater fish as the brook trout in the present study had a limited ability to acclimate to temperatures beyond 20°C.

Life through a wider scope: Brook Trout (Salvelinus fontinalis) exhibit similar aerobic scope across a broad temperature range.

Brook Trout (Salvelinus fontinalis) have been widely introduced throughout the world and are often considered as direct competitors with native salmonid species. Metabolic rate is one metric we can examine to improve our understanding of how well fish perform in different habitats, including across temperature gradients, as metabolism can be directly influenced by environmental temperatures in ectotherms. We estimated the standard metabolic rate, maximum metabolic rate, and aerobic scope of lab-reared juvenile Brook Trout (~1 year) using intermittent-flow respirometry across a range of temperatures (5-23 °C) likely experienced in the wild. We included a diurnal temperature cycle of ±1.5 °C for each treatment temperature to simulate temporal variation observed in natural waterbodies. Standard metabolic rate and maximum metabolic rate both increased with acclimation temperature before appearing to plateau around 20 °C, while mass specific aerobic scope was found to increase from a mean of 287.25 ± 13.03 mg O2·kg-1·h-1 at 5 °C to 384.85 ± 13.31 mg O2·kg-1·h-1 at 15 °C before dropping at higher temperatures. Although a slight peak was found at 15 °C, the generally flat thermal performance curve for aerobic scope suggests Brook Trout are capable of adjusting to a relatively wide range of thermal regimes, appearing to be eurythermal, or a thermal generalist, at least for salmonids. The ability of this population to maintain similar physiological performance across a wide range of temperatures may help explain why Brook Trout succeed in a variety of different thermal habitats.

Applying a gene-suite approach to examine the physiological status of wild-caught walleye (Sander vitreus).

Molecular techniques have been increasingly used in a conservation physiology framework to provide valuable information regarding the mechanisms underlying responses of wild organisms to environmental and anthropogenic stressors. In the present study, we developed a reference gill transcriptome for walleye (Sander vitreus), allowing us to pair a gene-suite approach (i.e. multiple genes across multiple cellular processes) with multivariate statistics to examine the physiological status of wild-caught walleye. For molecular analyses of wild fish, the gill is a useful target for conservation studies, not only because of its importance as an indicator of the physiological status of fish but also because it can be biopsied non-lethally. Walleye were non-lethally sampled following short- (~1.5 months) and long-term (~3.5 months) confinement in the Delta Marsh, which is located south of Lake Manitoba in Manitoba, Canada. Large-bodied walleye are confined in the Delta Marsh from late April to early August by exclusion screens used to protect the marsh from invasive common carp (Cyprinus carpio), exposing fish to potentially stressful water quality conditions. Principal components analysis revealed patterns of transcript abundance consistent with exposure of fish to increasingly high temperature and low oxygen conditions with longer holding in the marsh. For example, longer-term confinement in the marsh was associated with increases in the mRNA levels of heat shock proteins and a shift in the mRNA abundance of aerobic to anaerobic metabolic genes. Overall, the results of the present study suggest that walleye confined in the Delta Marsh may be exhibiting sub-lethal responses to high temperature and low oxygen conditions. These results provide valuable information for managers invested in mediating impacts to a local species of conservation concern. More broadly, we highlight the usefulness of pairing transcriptomic techniques with multivariate statistics to address potential confounding factors that can affect measured physiological responses of wild-caught fish.

Genomic signals found using RNA sequencing show signatures of selection and subtle population differentiation in walleye (Sander vitreus) in a large freshwater ecosystem.

RNA sequencing is an effective approach for studying aquatic species yielding both physiological and genomic data. However, its population genetic applications are not well-characterized. We investigate this possible role for RNA sequencing for population genomics in Lake Winnipeg, Manitoba, Canada, walleye (Sander vitreus). Lake Winnipeg walleye represent the largest component of the second-largest freshwater fishery in Canada. In the present study, large female walleye were sampled via nonlethal gill biopsy over two years at three spawning sites representing a latitudinal gradient in the lake. Genetic variation from sequenced mRNA was analyzed for neutral and adaptive markers to investigate population structure and possible adaptive variation. We find low population divergence (F ST = 0.0095), possible northward gene flow, and outlier loci that vary latitudinally in transcripts associated with cell membrane proteins and cytoskeletal function. These results indicate that Lake Winnipeg walleye may be effectively managed as a single demographically connected metapopulation with contributing subpopulations and suggest genomic differences possibly underlying observed phenotypic differences. Despite its high cost relative to other genotyping methods, RNA sequencing data can yield physiological in addition to genetic information discussed here. We therefore argue that it is useful for addressing diverse molecular questions in the conservation of freshwater species.

Sub-lethal temperature thresholds indicate acclimation and physiological limits in brook trout Salvelinus fontinalis.

The upper thermal tolerance of brook trout Salvelinus fontinalis was estimated using critical thermal maxima (CTmax ) experiments on fish acclimated to temperatures that span the species' thermal range (5-25°C). The CTmax increased with acclimation temperature but plateaued in fish acclimated to 20, 23 and 25°C. Plasma lactate was highest, and the hepato-somatic index (IH ) was lowest at 23 and 25°C, which suggests additional metabolic costs at those acclimation temperatures. The results suggest that there is a sub-lethal threshold between 20 and 23°C, beyond which the fish experience reduced physiological performance.

Hypoxia but not shy-bold phenotype mediates thermal preferences in a threatened freshwater fish, Notropis percobromus.

For ectothermic animals, ambient temperature strongly influences developmental growth rate and individual fitness. While many ectotherms live in environments that are spatially hetero-thermal, the coupling between behavioural phenotypes (e.g., shy or bold behaviour) and thermal preferences remains uncertain. Relative to shy counterparts, bolder phenotypes may exert higher preference for ambient temperatures that are closer to their thermal optimum, thereby accelerating development. In addition, ectotherms should select colder temperatures in low oxygen conditions (hypoxia) according to the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis. Using wild caught carmine shiner (Notropis percobromus), this study examined thermoregulatory behaviour in individuals exhibiting consistent behavioural phenotypes along the shy-bold continuum and between ecologically relevant normal oxygen concentration (normoxic) and hypoxic treatments. Furthermore, the behaviour observed in the laboratory was compared to environmental data from the natal stream. Results demonstrated that individual shy-bold behavioural phenotype was consistent before and after a simulated aerial predator attack, indicating consistency of behaviour across situations. Individual preferred and avoidance temperatures varied substantially, but were unrelated to shy-bold behavioural phenotypes. In contrast, individual preferred and maximum avoidance temperatures were significantly reduced in hypoxia, consistent with the OCLTT hypothesis. These findings might indicate suppressed development rates in hypoxia, not only by the limited oxygen for aerobic metabolism, but also by the preference for colder water in hypoxia. Furthermore, the tolerated thermal ranges were reduced in hypoxia. Using test conditions confirmed by field data, our study demonstrates the strong influence of oxygen availability on thermoregulatory behaviours and preferences in aquatic environments.

Standard metabolic rate models for Carmine Shiner (Notropis percobromus) and Common Shiner (Luxilus cornutus) across different temperature regimes.

Standard metabolic rates (SMR) were measured empirically for carmine shiner Notropis percobromus and common shiner Luxilus cornutus to develop SMR models that predict metabolic responses of each species under thermal conditions observed in the wild. SMR increased significantly with body mass and rising water temperature, ranging from 0.05 mg O2 h-1 at 10°C to 0.89 mg O2 h-1 at 20°C for N. percobromus weighing 0.6-2.5 g and from 0.11 mg O2 h-1 at 10°C to 0.98 mg O2 h-1 at 20°C for L. cornutus weighing 0.8-6.6 g. SMR models significantly differed between sympatric species on account of differences in model intercepts (RA) and temperature coefficients (RQ), however, the allometric relationships between mass and SMR did not significantly differ between species. Known distribution of N. percobromus and L. cornutus includes the Birch River located in Manitoba, Canada, where N. percobromus is listed as Endangered. Little is known about the physiology of N. percobromus or the species' ability to acclimate or adapt to different environmental conditions. While size differences between species contributed, in part, to differences in SMR predictions for Birch River populations, SMR trends (< 2 mg O2 h-1 ) for individuals weighing 1 g were similar for both species across daily temperatures. Respirometry experiments contributed to developing species-specific SMR models and inform on the effect of natural and anthropogenic stressors, namely water temperature, on the conservation of N. percobromus in this ecosystem.

Using the shuttlebox experimental design to determine temperature preference for juvenile Westslope Cutthroat Trout (Oncorhynchus clarkii lewisi).

Temperature preference for various fishes has often been used as a proxy of optimal temperature for growth and metabolism due to the ease of obtaining preferred temperature zones in laboratory experiments. Several laboratory designs and methods have been proposed to examine preferred temperature zones, however, differences between them (i.e. thermal gradients vs. static temperatures in chambers and duration of acclimation/experimental periods) have led to varying measurements, precluding comparisons between experiments, species and/or life-stages. Juvenile Westslope Cutthroat Trout (Oncorhynchus clarkii lewisi), a species listed as threatened in Alberta and of special concern in British Columbia, were tested in an automated shuttlebox experimental design (Loligo® Systems) to determine average and ranges of temperature preference (Tpref) and occupied temperatures. Previous lab studies suggested that Westslope Cutthroat Trout (WCT) prefer temperatures around 15°C, however, we found that average daytime Tpref for lab-reared juvenile WCT was substantially higher at 18.6°C, with occupied temperatures ranging between 11.9°C and 26.0°C throughout the duration of trials. This seems to indicate that despite constant lab-rearing conditions of 12°C, juvenile WCT may tolerate and even prefer warmer water temperatures. The duration of the acclimation period (1h, 12 h and 24 h) did not have an effect on Tpref, however, Tpref differed significantly for variable trial durations (12 h, 24 h and 36 h). A closer look at thermal trends throughout trials revealed that photoperiod significantly influenced Tpref, as nighttime temperature preference reached consistently 26°C. Collectively, these results suggest that shuttlebox experiments on WCT need to take into account the photoperiod, as behaviour may drive Tpref more so than the duration of acclimation periods.

Environmental calcium and variation in yolk sac size influence swimming performance in larval lake sturgeon (Acipenser fulvescens).

In many animal species, performance in the early life stages strongly affects recruitment to the adult population; however, factors that influence early life history stages are often the least understood. This is particularly relevant for lake sturgeon, Acipenser fulvescens, living in areas where environmental calcium concentrations are declining, partly due to anthropogenic activity. As calcium is important for muscle contraction and fatigue resistance, declining calcium levels could constrain swimming performance. Similarly, swimming performance could be influenced by variation in yolk sac volume, because the yolk sac is likely to affect drag forces during swimming. Testing swimming performance of larval A. fulvescens reared in four different calcium treatments spanning the range of 4-132 mg l-1 [Ca2+], this study found no treatment effects on the sprint swimming speed. A novel test of volitional swimming performance, however, revealed reduced swimming performance in the low calcium environment. Specifically, volitionally swimming larvae covered a shorter distance before swimming cessation in the low calcium environment compared with the other treatments. Moreover, sprint swimming speed in larvae with a large yolk sac was significantly slower than in larvae with a small yolk sac, regardless of body length variation. Thus, elevated maternal allocation (i.e. more yolk) was associated with reduced swimming performance. Data suggest that larvae in low calcium environments or with a large yolk sac exhibit reduced swimming performance and could be more susceptible to predation or premature downstream drift. Our study reveals how environmental factors and phenotypic variation influence locomotor performance in a larval fish.

Climate change risks, extinction debt, and conservation implications for a threatened freshwater fish: Carmine shiner (Notropis percobromus).

Climate change is affecting many freshwater species, particularly fishes. Predictions of future climate change suggest large and deleterious effects on species with narrow dispersal abilities due to limited hydrological connectivity. In turn, this creates the potential for population isolation in thermally unsuitable habitats, leading to physiological stress, species declines or possible extirpation. The current extent of many freshwater fish species' spatio-temporal distribution patterns and their sensitivity to thermal impacts from climate change - critical information for conservation planning - are often unknown. Carmine shiner (Notropis percobromus) is an ecologically important species listed as threatened or imperilled nationally (Canada) and regionally (South Dakota, United States) due to its restricted range and sensitivity to water quality and temperature. This research aimed to determine the current distribution and spatio-temporal variability in projected suitable habitat for Carmine shiner using niche-based modeling approaches (MaxEnt, BIOCLIM, and DOMAIN models). Statistically downscaled, bias-corrected Global Circulation Models (GCMs) data was used to model the distribution of Carmine shiner in central North America for the period of 2041-2060 (2050s). Maximum mean July temperature and temperature variability were the main factors in determining Carmine shiner distribution. Patterns of projected habitat change by the 2050s suggest the spatial extent of the current distribution of Carmine shiner would shift north, with >50% of the current distribution changing with future projections based on two Representative Concentrations Pathways for CO2 emissions. Whereas the southern extent of the distribution would become unsuitable for Carmine shiner, suitable habitats are predicted to become available further north, if accessible. Importantly, the majority of habitat gains for Carmine shiner would be in areas currently inaccessible due to dispersal limitations, suggesting current populations may face an extinction debt within the next half century. These results provide evidence that Carmine shiner may be highly vulnerable to a warming climate and suggest that management actions - such as assisted migration - may be needed to mitigate impacts from climate change and ensure the long-term persistence of the species.

Effect of nanosilver on metabolism in rainbow trout (Oncorhynchus mykiss): An investigation using different respirometric approaches.

Nanosilver (nAg) has been incorporated into many consumer products, including clothing and washing machines, because of its antimicrobial properties. Consequently, the potential for its release into aquatic environments is of significant concern. Documented toxic effects on fish include altered gene expression, gill damage, and impaired gas exchange, as well as mortality at high nAg concentrations. The present study reports the effects of nAg on the metabolism of rainbow trout (Oncorhynchus mykiss). Fish were exposed to environmentally relevant concentrations (0.28 ± 0.02 µg/L) and higher (47.60 ± 5.13 µg/L) for 28 d, after which their standard metabolic rate (SMR), forced maximum metabolic rate (MMRf ), and spontaneous maximum metabolic rate (MMRs ) were measured. There was no effect observed in SMR, MMRf , or MMRs , suggesting that nAg is unlikely to directly affect fish metabolism. On average, MMRs tended to be greater than MMRf , and most MMRs occurred when room lighting increased. The timing of MMRf chase protocols was found to affect both MMRf and SMR estimates, in that chasing fish before respirometric experiments caused higher MMRf estimates and lower SMR estimates. Although compounded effects involving nAg and other environmental stressors remain unknown, the present study indicates that the tested range of nAg is unlikely to constrain fish metabolism. Environ Toxicol Chem 2017;36:2722-2729. © 2017 SETAC.

Development and testing of a simple field-based intermittent-flow respirometry system for riverine fishes.

By understanding range-wide intraspecific variation in metabolic rate we can better understand how organisms have adapted to their environment. However, methods to quantify metabolic rate of fishes from remote areas or those that cannot be brought back to the laboratory because of imperilment status are lacking. Consequently, practical and reliable field-based methods are needed. To address this need, we developed a simple yet robust intermittent-flow respirometry system, adapted from a design commonly used in the laboratory that is readily suited for field use. Standard metabolic rate (SMR), maximum metabolic rate (MMR) and aerobic scope (AS) estimates were obtained from juvenile lake trout (Salvelinus namaycush) and brook trout (Salvelinus fontinalis) using both field- and laboratory-based systems. Whole-fish SMR, MMR and AS estimates from the field and laboratory methods did not differ from one another (ANCOVA and LMM: all P > 0.05) for either species and were comparable to estimates previously reported. Our field setup is a simpler system than the conventional laboratory-based system that requires less power and equipment to operate, yet still offers users the ability to: (1) acclimate fish to the respirometry chamber; (2) measure oxygen consumption during a shorter period (1 h), which yield metabolic rate estimates comparable to systems that take measurements over longer periods; and (3) take repeated oxygen consumption measurements with manual user-defined flush and measurement phase routines. Developing practical and reliable field respirometry methods, as demonstrated here, is important if we wish to improve our ability to predict how imperiled species will respond to changes in their environment. Such knowledge is critical for informing conservation strategies.

Effect of nanosilver on cortisol release and morphometrics in rainbow trout (Oncorhynchus mykiss).

Nanosilver (nAg) is a nanoparticle commonly incorporated into consumer products for its antimicrobial properties that has been detected in aquatic environments. Toxic effects of nAg on fish have been observed, and nAg may induce a stress response in fish in the form of increased blood plasma cortisol. Effects of nAg exposure on rainbow trout (Oncorhynchus mykiss) were investigated over a 28-d period using blood plasma cortisol concentrations as an indicator of stress. Several morphometric measures (growth, Fulton's condition factor, and hepatosomatic index [HSI]) were also taken during the experiment to investigate potential whole-body effects of exposure, and concentrations of nAg in fish muscle tissue were measured. Fish were exposed to environmentally relevant (average 0.28 µg/L) and higher (average 47.60 µg/L) exposure concentrations of nAg. The results showed a significant increase in blood plasma cortisol for both exposure treatments. A significant effect on HSI by treatment dependent on exposure time was also observed, although no obvious trend was detected, whereas other morphometric measures were not affected by nAg exposure. In addition, Ag was detected in fish muscle tissue. The results indicate that although nAg did engage the stress response in fish, it did not affect growth or condition under the experimental conditions and time frame investigated. Environ Toxicol Chem 2017;36:1606-1613. © 2016 SETAC.

Estimates of metabolic rate and major constituents of metabolic demand in fishes under field conditions: Methods, proxies, and new perspectives.

Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as well as the role of species in their ecosystem. Despite the ecological and commercial importance of fishes, there are currently no widely adopted means of measuring field metabolic rate in fishes. The lack of recognized methods is in part due to the logistical difficulties of measuring metabolic rates in free swimming fishes. However, further development and refinement of techniques applicable for field-based studies on free swimming animals would greatly enhance the capacity to study fish under environmentally relevant conditions. In an effort to foster discussion in this area, from field ecologists to biochemists alike, we review aspects of energy metabolism and give details on approaches that have been used to estimate energetic parameters in fishes. In some cases, the techniques have been applied to field conditions; while in others, the methods have been primarily used on laboratory held fishes but should be applicable, with validation, to fishes in their natural environment. Limitations, experimental considerations and caveats of these measurements and the study of metabolism in wild fishes in general are also discussed. Potential novel approaches to FMR estimates are also presented for consideration. The innovation of methods for measuring field metabolic rate in free-ranging wild fish would revolutionize the study of physiological ecology.

Evidence of circadian rhythm, oxygen regulation capacity, metabolic repeatability and positive correlations between forced and spontaneous maximal metabolic rates in lake sturgeon Acipenser fulvescens.

Animal metabolic rate is variable and may be affected by endogenous and exogenous factors, but such relationships remain poorly understood in many primitive fishes, including members of the family Acipenseridae (sturgeons). Using juvenile lake sturgeon (Acipenser fulvescens), the objective of this study was to test four hypotheses: 1) A. fulvescens exhibits a circadian rhythm influencing metabolic rate and behaviour; 2) A. fulvescens has the capacity to regulate metabolic rate when exposed to environmental hypoxia; 3) measurements of forced maximum metabolic rate (MMR(F)) are repeatable in individual fish; and 4) MMR(F) correlates positively with spontaneous maximum metabolic rate (MMR(S)). Metabolic rates were measured using intermittent flow respirometry, and a standard chase protocol was employed to elicit MMR(F). Trials lasting 24 h were used to measure standard metabolic rate (SMR) and MMR(S). Repeatability and correlations between MMR(F) and MMR(S) were analyzed using residual body mass corrected values. Results revealed that A. fulvescens exhibit a circadian rhythm in metabolic rate, with metabolism peaking at dawn. SMR was unaffected by hypoxia (30% air saturation (O(2sat))), demonstrating oxygen regulation. In contrast, MMR(F) was affected by hypoxia and decreased across the range from 100% O(2sat) to 70% O(2sat). MMR(F) was repeatable in individual fish, and MMR(F) correlated positively with MMR(S), but the relationships between MMR(F) and MMR(S) were only revealed in fish exposed to hypoxia or 24 h constant light (i.e. environmental stressor). Our study provides evidence that the physiology of A. fulvescens is influenced by a circadian rhythm and suggests that A. fulvescens is an oxygen regulator, like most teleost fish. Finally, metabolic repeatability and positive correlations between MMR(F) and MMR(S) support the conjecture that MMR(F) represents a measure of organism performance that could be a target of natural selection.