Interindividual behavioural variation in response to elevated CO2 predicts mRNA transcript abundance of genes related to acid-base regulation in medaka (Oryzias latipes).

Rising carbon dioxide (CO2) in aquatic ecosystems due to climate change is a challenge for aquatic ectotherms. We examined whether interindividual variation in behavioural responses to CO2 could predict how a teleost fish would respond to elevated CO2 for multiple phenotypic and molecular traits. To this end, we first quantified behavioural responses of individuals exposed to acute elevated CO2, and used these to assign individuals as either high or low responders relative to the population mean. Subsequently, we exposed both high and low responders to elevated CO2 for 6 weeks and quantified the effect on body condition, behaviour, and mRNA transcript responses of gill and liver genes associated with relevant physiological processes. Generally, we found few relationships between the phenotypic groups and body condition and behaviour following the CO2 exposure period; however, stark differences between the phenotypic groups with respect to gene transcripts from each tissue related to various processes were found, mostly independent of CO2 exposure. The most pronounced changes were in the gill transcripts related to acid-base regulation, suggesting that the observed behavioural variation used to assign fish to phenotypic groups may have an underlying molecular origin. Should the link between behaviour and gene transcripts be shown to have a fitness advantage and be maintained across generations, interindividual variation in behavioural responses to acute CO2 exposure may be a viable and non-invasive tool to predict future population responses to elevated aquatic CO2.

Are the effects of catch-and-release angling evident in changes to mRNA abundances related to metabolism, acid-base regulation and stress in lake trout (Salvelinus namaycush) gills?

Catch-and-release (C&R) angling is a conservation-oriented practice intended to reduce the impact recreational angling has on fish populations. Even though most recreationally angled fish are released, little is known about how C&R angling impacts fish at the cellular or tissue level. As the first to explore the impacts of C&R angling on mRNA abundances, our study aimed to identify how the stress of angling influenced metabolism, acid-base regulation and cellular stress in the gills of lake trout (Salvelinus namaycush). Because gills are responsible for metabolic gas exchange, are crucial sites of acid-base homeostasis and respond to stressors quickly, we hypothesized that the relative mRNA abundance of genes related to these three physiological processes would be altered after angling. We took gill samples of live lake trout at 0, 2 or 48 h after fish were angled by rod and reel, and then used quantitative PCR (qPCR) to measure the relative abundance of nine candidate mRNA transcripts. Heat shock protein 70 (hsp70) mRNA levels significantly increased over 5-fold 2 h after angling, indicating a potential activation of a cytoprotective response. However, contrary to our hypothesis, we observed no change in the relative mRNA abundance of genes related to metabolism or acid-base regulation in response to C&R angling within a 48-h period. As C&R angling can negatively impact fish populations, further use of transcript-level studies will allow us to understand the impact C&R has on specific tissues and improve our knowledge of how C&R influences overall fish health.

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.

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.

Physiological status of silver carp (Hypophthalmichthys molitrix) in the Illinois River: An assessment of fish at the leading edge of the invasion front.

Silver carp (Hypophthalmichthys molitrix) are invasive to North America, and their range has expanded within the Mississippi River Basin, seemingly unchecked, since their introduction in the late 1970s, with the exception of the upper reaches of the Illinois River. With the imminent threat of their movement into the Great Lakes, the goal of the present study was to assess whether differences in the physiological status between silver carp at the leading edge of their invasion front and core population sites could explain their lack of expansion upstream toward Lake Michigan over the past decade. A transcriptomic approach using RNA sequencing and analysis of plasma variables were used to quantify differences among fish at the leading edge and two downstream core population sites. Leading-edge fish exhibited upregulation of genes associated with xenobiotic defense (e.g., ATP-binding cassette C1 [abcc1], abcc2, abcc6), decreased cell integrity (i.e., macroautophagy and apoptosis; autophagy-related protein 9A [atg9a], caspase 3b [casp3b]), and cholesterol metabolism (e.g., abca1, apolipoprotein A1 [apoa1], sterol O-acyltransferase [soat1]) and downregulation of genes associated with DNA repair (e.g., tumor suppressor p53-binding protein 1 [tp53bp1]) compared to core population sites. Transcriptomic profiles of leading-edge fish were consistent with fish inhabiting a polluted environment and suggest that poorer water quality conditions upstream of the leading edge may represent a non-permanent barrier to silver carp range expansion. The present study provides potential molecular targets for monitoring the physiological status of silver carp over time and in response to future improvements in water quality upstream of their leading edge.

Hot and bothered: effects of elevated Pco2 and temperature on juvenile freshwater mussels.

Multiple environmental stressors may interact in complex ways to exceed or diminish the impacts of individual stressors. In the present study, the interactive effects of two ecologically relevant stressors [increased temperature and partial pressure of carbon dioxide (Pco2)] were assessed for freshwater mussels, a group of organisms that are among the most sensitive and rapidly declining worldwide. The individual and combined effects of elevated temperature (22°C-34°C) and Pco2 (~230, 58,000 µatm) on juvenile Lampsilis siliquoidea were quantified over a 5- or 14-day period, during which physiological and whole animal responses were measured. Exposure to elevated temperature induced a series of physiological responses, including an increase in oxygen consumption rates following 5 days of exposure at 31°C and an increase in carbonic anhydrase ( ca) and heat shock protein 70 mRNA levels following 14 days of exposure at 28°C and 34°C, respectively. Treatment with elevated Pco2 activated acid-base regulatory responses including increases in CA and Na+-K+-ATPase activity and a novel mechanism for acid-base regulation during Pco2 exposure in freshwater mussels was proposed. Thermal and CO2 stressors also interacted such that responses to the thermal stressor were diminished in mussels exposed to elevated Pco2, resulting in the greatest level of mortality. Additionally, larger mussels were more likely to survive treatment with elevated Pco2 and/or temperature. Together, exposure to elevated Pco2 may compromise the ability of juvenile freshwater mussels to respond to additional stressors, such as increased temperatures, highlighting the importance of considering not only the individual but also the interactive effects of multiple environmental stressors.

Chronic exposure of a freshwater mussel to elevated pCO2 : Effects on the control of biomineralization and ion-regulatory responses.

Freshwater mussels may be exposed to elevations in mean partial pressure of carbon dioxide (pCO2 ) caused by both natural and anthropogenic factors. The goal of the present study was to assess the effects of a 28-d elevation in pCO2 at 15 000 and 50 000 µatm on processes associated with biomineralization, ion regulation, and cellular stress in adult Lampsilis siliquoidea (Barnes, 1823). In addition, the capacity for mussels to compensate for acid-base disturbances experienced after exposure to elevated pCO2 was assessed over a 14-d recovery period. Overall, exposure to 50 000 µatm pCO2 had more pronounced physiological consequences compared with 15 000 µatm pCO2 . Over the first 7 d of exposure to 50 000 µatm pCO2 , the mRNA abundance of chitin synthase (cs), calmodulin (cam), and calmodulin-like protein (calp) were significantly affected, suggesting that shell formation and integrity may be altered during pCO2 exposure. After the removal of the pCO2 treatment, mussels may compensate for the acid-base and ion disturbances experienced during pCO2 exposure, and transcript levels of some regulators of biomineralization (carbonic anhydrase [ca], cs, cam, calp) as well as ion regulation (na+ -k+ -ATPase [nka]) were modulated. Effects of elevated pCO2 on heat shock protein 70 (hsp70) were limited in the present study. Overall, adult L. siliquoidea appeared to regulate factors associated with the control of biomineralization and ion regulation during and/or after the removal of pCO2 exposure. Environ Toxicol Chem 2018;37:538-550. © 2017 SETAC.

Valve movement of three species of North American freshwater mussels exposed to elevated carbon dioxide.

Freshwater mussels are at-risk taxa and may be exposed to high levels of carbon dioxide (CO2) because of the potential use of CO2 to control the movement of invasive aquatic fish species. One potential behavioral response to a change in the partial pressure of CO2 (pCO2) may be altered valve movement. In this study, three species of mussels were fitted with modified sensors and exposed to two regimes of pCO2 to define thresholds of impaired valve movement. The first experiment demonstrated that Pyganodon grandis were much more tolerant to rising pCO2 relative to Lampsilis siliquoidea (acute closure at ∼200,000 µatm in comparison to ∼80,000 µatm). The second experiment consisted of monitoring mussels for 6 days and exposing them to elevated pCO2 (∼70,000 µatm) over a 2-day period. During exposure to high pCO2, Lampsilis cardium were open for nearly the entire high pCO2 period. Conversely, P. grandis were closed for most of the period following exposure to high pCO2. For L. siliquoidea, the number of closures decreased nearly 40-fold during high pCO2. The valve movement responses observed suggest species differences, and exposure to elevated pCO2 requires a reactive response.

Responses to elevated CO2 exposure in a freshwater mussel, Fusconaia flava.

Freshwater mussels are some of the most imperiled species in North America and are particularly susceptible to environmental change. One environmental disturbance that mussels may encounter that remains understudied is an increase in the partial pressure of CO2 (pCO2). The present study quantified the impacts of acute (6 h) and chronic (up to 32 days) exposures to elevated pCO2 on genes associated with shell formation (chitin synthase; cs) and the stress response (heat shock protein 70; hsp70) in Fusconaia flava. Oxygen consumption (MO2) was also assessed over the chronic CO2 exposure period. Although mussels exhibited an increase in cs following an acute exposure to elevated pCO2, long-term exposure resulted in a decrease in cs mRNA abundance, suggesting that mussels may invest less in shell formation during chronic exposure to elevated pCO2. In response to an acute elevation in pCO2, mussels increased hsp70 mRNA abundance in mantle and adductor muscle and a similar increase was observed in the gill and adductor muscle in response to a chronic elevation in pCO2. A chronic elevation in pCO2 also increased mussel MO2. This overall increase in hsp70 mRNA levels and MO2 in F. flava indicates that exposure to elevated pCO2 initiates activation of the general stress response and an increased energy demand. Together, the results of the present study suggest that freshwater mussels respond to elevated pCO2 by increasing processes necessary to 'deal with' the stressor and, over the long-term, may reduce their investment in non-essential processes such as shell growth.

The response of two species of unionid mussels to extended exposure to elevated carbon dioxide.

Changes in environmental conditions can act as stressors, with potential consequences for the health and fitness of organisms. Rising levels of carbon dioxide (CO2) is one potential environmental stressor that is occurring more frequently in the environment and can be a stressor for aquatic organisms. In this study, the physiological responses of two species of unionid mussel, Lampsilis siliquoidea and Amblema plicata, were assessed in response to exposure to two levels of elevated partial pressure of CO2 (pCO2) (~20,000 and ~55,000µatm) over a 28d period, followed by a subsequent 14d recovery period. Observations were consistent with responses associated with respiratory acidosis, as demonstrated by changes in hemolymph HCO3(-), Ca(2+), Cl(-), and Na(2+). Both species exposed to elevated pCO2 had elevated hemolymph HCO3(-) during the pCO2 treatment period compared to control mussels, but recovered once pCO2 was removed. Similarly, both species had elevated hemolymph Na(+) during exposure to elevated pCO2, and this returned to control levels for A. plicata but remained elevated for L. siliquoidea once the pCO2 stimuli was removed. Changes in hemolymph Ca(2+) and Cl(-) in response to elevated pCO2 were also observed, but these changes were species-specific. Additional physiological responses to elevated pCO2 (e.g., changes in hemolymph glucose and Mg(2+)) were consistent with a stress response in both species. This study demonstrates the importance of considering inter-specific differences in the response of organisms to stress, and also that responses to elevated pCO2 may be transient and can recover once the stress is removed.

Programming of the hypothalamic-pituitary-interrenal axis by maternal social status in zebrafish (Danio rerio).

The present study examined the effects of maternal social status, with subordinate status being a chronic stressor, on development and activity of the stress axis in zebrafish embryos and larvae. Female zebrafish were confined in pairs for 48 h to establish dominant/subordinate hierarchies; their offspring were reared to 144 h post-fertilization (hpf) and sampled at five time points over development. No differences were detected in maternal cortisol contribution, which is thought to be an important programmer of offspring phenotype. However, once zebrafish offspring began to synthesize cortisol de novo (48 hpf), larvae of dominant females exhibited significantly lower baseline cortisol levels than offspring of subordinate females. These lower cortisol levels may reflect reduced hypothalamic-pituitary-interrenal (HPI) axis activity, because corticotropin-releasing factor (crf) and cytochrome p450 side chain cleavage enzyme (p450scc) mRNA levels also were lower in larvae from dominant females. Moreover, baseline mRNA levels of HPI axis genes continued to be affected by maternal social status beyond 48 hpf. At 144 hpf, stress-induced cortisol levels were significantly lower in offspring of subordinate females. These results suggest programming of stress axis function in zebrafish offspring by maternal social status, emphasizing the importance of maternal environment and experience on offspring stress axis activity.

Freshwater biota and rising pCO2?

Rising atmospheric carbon dioxide (CO2) has caused a suite of environmental issues, however, little is known about how the partial pressure of CO2 (pCO2) in freshwater will be affected by climate change. Freshwater pCO2 varies across systems and is controlled by a diverse array of factors, making it difficult to make predictions about future levels of pCO2. Recent evidence suggests that increasing levels of atmospheric CO2 may directly increase freshwater pCO2 levels in lakes, but rising atmospheric CO2 may also indirectly impact freshwater pCO2 levels in a variety of systems by affecting other contributing factors such as soil respiration, terrestrial productivity and climate regimes. Although future freshwater pCO2 levels remain uncertain, studies have considered the potential impacts of changes to pCO2 levels on freshwater biota. Studies to date have focused on impacts of elevated pCO2 on plankton and macrophytes, and have shown that phytoplankton nutritional quality is reduced, plankton community structure is altered, photosynthesis rates increase and macrophyte distribution shifts with increasing pCO2. However, a number of key knowledge gaps remain and gaining a better understanding of how freshwater pCO2 levels are regulated and how these levels may impact biota, will be important for predicting future responses to climate change.

Physiological responses of three species of unionid mussels to intermittent exposure to elevated carbon dioxide.

Freshwater systems are at risk owing to increasing carbon dioxide (CO2) levels, and one of the possible reasons for these elevations is the deployment of non-physical fish barriers to prevent invasive fish movements. Carbon dioxide barriers have the potential to create short, chronic and intermittent exposures of CO2 for surrounding freshwater biota. Although intermittent exposures to a stressor may be more ecologically relevant, the majority of laboratory tests use chronic or short-term time periods to determine how organisms will respond to an environmental stressor. Measurements of the physiological responses of three species of unionid mussel, giant floaters (Pyganodon grandis), threeridge (Amblema plicata) and plain pocketbook (Lampsilis cardium), exposed to control pCO2 (~1000 µatm) or intermittent conditions of pCO2 (ranging from ~1000 to ~55 000 µatm) 12 times per day over a 28 day period were gathered. There was no indication of recovery in the physiological responses of mussels between applications of CO2, suggesting that the recovery time between CO2 pulses (1.5 h) was not sufficient for recovery from the CO2 exposure period (0.5 h). Observations of acid-base and stress responses were consistent with what has been observed in chronic studies of freshwater mussels exposed to elevated pCO2 (i.e. elevations in HCO3-, Ca2+, Na+ and glucose, and decreases in Mg2+ and Cl-). However, species differences were observed across almost all variables measured, which emphasizes the need for multispecies studies.

Linking Landscape-Scale Disturbances to Stress and Condition of Fish: Implications for Restoration and Conservation.

Humans have dramatically altered landscapes as a result of urban and agricultural development, which has led to decreases in the quality and quantity of habitats for animals. This is particularly the case for freshwater fish that reside in fluvial systems, given that changes to adjacent lands have direct impacts on the structure and function of watersheds. Because choices of habitat have physiological consequences for organisms, animals that occupy sub-optimal habitats may experience increased expenditure of energy or homeostatic overload that can cause negative outcomes for individuals and populations. With the imperiled and threatened status of many freshwater fish, there is a critical need to define relationships between land use, quality of the habitat, and physiological performance for resident fish as an aid to restoration and management. Here, we synthesize existing literature to relate variation in land use at the scale of watersheds to the physiological status of resident fish. This examination revealed that landscape-level disturbances can influence a host of physiological properties of resident fishes, ranging from cellular and genomic levels to the hormonal and whole-animal levels. More importantly, these physiological responses have been integrated into traditional field-based monitoring protocols to provide a mechanistic understanding of how organisms interact with their environment, and to enhance restoration. We also generated a conceptual model that provides a basis for relating landscape-level changes to physiological responses in fish. We conclude that physiological sampling of resident fish has the potential to assess the effects of landscape-scale disturbances on freshwater fish and to enhance restoration and conservation.

Use of portable blood physiology point-of-care devices for basic and applied research on vertebrates: a review.

Non-human vertebrate blood is commonly collected and assayed for a variety of applications, including veterinary diagnostics and physiological research. Small, often non-lethal samples enable the assessment and monitoring of the physiological state and health of the individual. Traditionally, studies that rely on blood physiology have focused on captive animals or, in studies conducted in remote settings, have required the preservation and transport of samples for later analysis. In either situation, large, laboratory-bound equipment and traditional assays and analytical protocols are required. The use of point-of-care (POC) devices to measure various secondary blood physiological parameters, such as metabolites, blood gases and ions, has become increasingly popular recently, due to immediate results and their portability, which allows the freedom to study organisms in the wild. Here, we review the current uses of POC devices and their applicability to basic and applied studies on a variety of non-domesticated species. We located 79 individual studies that focused on non-domesticated vertebrates, including validation and application of POC tools. Studies focused on a wide spectrum of taxa, including mammals, birds and herptiles, although the majority of studies focused on fish, and typical variables measured included blood glucose, lactate and pH. We found that calibrations for species-specific blood physiology values are necessary, because ranges can vary within and among taxa and are sometimes outside the measurable range of the devices. In addition, although POC devices are portable and robust, most require durable cases, they are seldom waterproof/water-resistant, and factors such as humidity and temperature can affect the performance of the device. Overall, most studies concluded that POC devices are suitable alternatives to traditional laboratory devices and eliminate the need for transport of samples; however, there is a need for greater emphasis on rigorous calibration and validation of these units and appreciation of their limitations.

Modulation of hypothalamic-pituitary-interrenal axis function by social status in rainbow trout.

Juvenile rainbow trout (Oncorhynchus mykiss) form stable dominance hierarchies when confined in pairs. These hierarchies are driven by aggressive competition over limited resources and result in one fish becoming dominant over the other. An important indicator of low social status is sustained elevation of circulating cortisol levels as a result of chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis. In the present study it was hypothesized that social status modulates the expression of key proteins involved in the functioning of the HPI axis. Cortisol treatment and fasting were used to assess whether these characteristics seen in subordinate fish also affected HPI axis function. Social status modulated plasma adrenocorticotropic hormone (ACTH) levels, cortisol synthesis, and liver glucocorticoid receptor (GR) expression. Plasma ACTH levels were lower by approximately 2-fold in subordinate and cortisol-treated fish, consistent with a negative feedback role for cortisol in modulating HPI axis function. Although cortisol-treated fish exhibited differences in corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP) mRNA relative abundances in the preoptic area and telencephalon, respectively, no effect of social status on CRF or CRF-BP was detected. Head kidney melanocortin 2 receptor (MC2R) mRNA relative levels were unaffected by social status, while mRNA relative abundances of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) enzyme were elevated in dominant fish. Liver GR2 mRNA and total GR protein levels in subordinate fish were lower than control values by approximately 2-fold. In conclusion, social status modulated the functioning of the HPI axis in rainbow trout. Our results suggest altered cortisol dynamics and reduced target tissue response to this steroid in subordinate fish, while the higher transcript levels for steroid biosynthesis in dominant fish leads us to propose an adaptive role for responding to subsequent stressors.