Integrated organismal responses induced by projected levels of CO2 and temperature exposures in the early life stages of lake sturgeon.

Atmospheric CO2 and temperature are rising concurrently, and may have profound impacts on the transcriptional, physiological and behavioural responses of aquatic organisms. Further, spring snowmelt may cause transient increases of pCO2 in many freshwater systems. We examined the behavioural, physiological and transcriptomic responses of an ancient fish, the lake sturgeon (Acipenser fulvescens) to projected levels of warming and pCO2 during its most vulnerable period of life, the first year. Specifically, larval fish were raised in either low (16°C) or high (22°C) temperature, and/or low (1000 µatm) or high (2500 µatm) pCO2 in a crossed experimental design over approximately 8 months. Following overwintering, lake sturgeon were exposed to a transient increase in pCO2 of 10,000 µatm, simulating a spring melt based on data in freshwater systems. Transcriptional analyses revealed potential connections to otolith formation and reduced growth in fish exposed to high pCO2 and temperature in combination. Network analyses of differential gene expression revealed different biological processes among the different treatments on the edges of transcriptional networks. Na+/K+-ATPase activity increased in fish not exposed to elevated pCO2 during development, and mRNA abundance of the ß subunit was most strongly predictive of enzyme activity. Behavioural assays revealed a decrease in total activity following an acute CO2 exposure. These results demonstrate compensatory and compounding mechanisms of pCO2 and warming dependent on developmental conditions in lake sturgeon. Conserved elements of the cellular stress response across all organisms provide key information for how other freshwater organisms may respond to future climate change.

Short-term exposure to high pCO2 leads to decreased branchial cytochrome C oxidase activity in the presence of octopamine in a decapod.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO2 exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO2 accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO3- observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO2 exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO2 exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO2 exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation. SUMMARY STATEMENT: Acid-base compensation upon short-term high pCO2 exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO3-, and regulation of mitochondrial complex IV (cytochrome C oxidase).

Comparison of metabolic rate between two genetically distinct populations of lake sturgeon.

Environmental temperatures differ across latitudes in the temperate zone, with relatively lower summer and fall temperatures in the north leading to a shorter growing season prior to winter. As an adaptive response, during early life stages, fish in northern latitudes may grow faster than their conspecifics in southern latitudes, which potentially manifests as different allometric relationships between body mass and metabolic rate. In the present study, we examined if population or year class had an effect on the variation of metabolic rate and metabolic scaling of age-0 lake sturgeon (Acipenser fulvescens) by examining these traits in both a northern (Nelson River) and a southern (Winnipeg River) population. We compiled 6 years of data that used intermittent flow respirometry to measure metabolic rate within the first year of life for developing sturgeon that were raised in the same environment at 16°C. We then used a Bayesian modeling approach to examine the impacts of population and year class on metabolic rate and mass-scaling of metabolic rate. Despite previous reports of genetic differences between populations, our results showed that there were no significant differences in standard metabolic rate, routine metabolic rate, maximum metabolic rate, and metabolic scaling between the two geographically separated populations at a temperature of 16°C. Our analysis implied that the lack of metabolic differences between populations could be due to family effects/parental contribution, or the rearing temperature used in the study. The present research provided insights for conservation and reintroduction strategies for these populations of lake sturgeon, which are endangered or threatened across most of their natural range.

Elevated temperatures dampen the innate immune capacity of developing lake sturgeon (Acipenser fulvescens).

Chronic exposure to high temperatures may leave freshwater fishes vulnerable to opportunistic pathogens, particularly during early life stages. Lake sturgeon, Acipenser fulvescens, populations within the northern expanse of their range in Manitoba, Canada, may be susceptible to high temperature stress and pathogenic infection. We acclimated developing lake sturgeon for 22 days to two ecologically relevant, summer temperatures (16 and 20°C). Individuals from both acclimation treatments were then exposed to 0, 30 and 60 µg ml-1 bacterial lipopolysaccharides (endotoxins), as an immune stimulus, for 48 h and sampled 4 and 48 h during trial exposures and following a 7 day recovery period. We then measured whole-body transcriptional (mRNA) responses involved in the innate immune, stress and fatty acid responses following acute exposure to the bacterial endotoxins. Data revealed that overall levels of mRNA transcript abundance were higher in 20°C-reared sturgeon under control conditions. However, following exposure to a bacterial stimulus, lake sturgeon acclimated to 16°C produced a more robust and persistent transcriptional response with higher mRNA transcript abundance across innate immune, stress and fatty acid responses than their 20°C-acclimated counterparts. Additional whole-animal performance metrics (critical thermal maximum, metabolic rate, cortisol concentration and whole-body and mucosal lysozyme activity) demonstrated acclimation-specific responses, indicating compromised metabolic, stress and enzymatic capacity following the initiation of immune-related responses. Our study showed that acclimation to 20°C during early development impaired the immune capacity of developing lake sturgeon as well as the activation of molecular pathways involved in the immune, stress and fatty acid responses. The present study highlights the effects of ecologically relevant, chronic thermal stress on seasonal pathogen susceptibility in this endangered species.

Long-term effects of temperature during early life on growth and fatty acid metabolism in age-0 Lake Sturgeon (Acipenser fulvescens).

Environmental temperature during early life may have prolonged effects on growth and fatty acid metabolism, which could strongly influence overwintering survival in the first year of life for temperate-zone fish. In the present study, we examined how temperature during early life history might influence growth performance and fatty acid metabolism in age-0 Lake Sturgeon (Acipenser fulvescens) when exposed to cold temperatures at later stages. Fish were initially at 16 °C and subsequently held at 16 °C or 20 °C for 60 days beginning at 34 days post fertilization (dpf). Then, all fish were subsequently raised at the same temperature of 16 °C until the onset of cold conditioning at 158 dpf where temperature was gradually decreased to 3.5 °C and remained there for two weeks. Samples were collected before (151 dpf) and after cold conditioning (199 dpf) to measure total length, body mass, whole body metabolic rate, fatty acid profile in phospholipids and triglycerides and mRNA expression of genes associated with fatty acid desaturation, elongation and ß-oxidation. Results revealed that before cold conditioning, total length and body mass did not differ between temperature groups, but fish raised at 20 °C showed a lower condition factor. During the cold conditioning, only fish raised at 16 °C grew significantly longer and heavier. There was no difference in metabolic rates between treatments. Significant increases in total monounsaturated fatty acids with decreases in total saturated fatty acids were identified in phospholipids and triglycerides in both temperature groups after the cold conditioning; however, the 20 °C group did not significantly increase levels of gene expression associated with fatty acid desaturation (SCD and FADS1) whereas the 16 °C group did. Our results suggest that thermal experience during early life may influence overwintering survival of age-0 Lake Sturgeon.

Effects of single and dual-stressor elevation of environmental temperature and PCO2 on metabolism and acid-base regulation in the Louisiana red swamp crayfish, Procambarus clarkii.

Elevation of temperature and CO2 levels within the world's aquatic environments is expected to cause numerous physiological challenges to their inhabitants. While effects on marine ecosystems have been well studied, freshwater ecosystems have rarely been examined using a dual-stressor approach leaving our understanding of its inhabitants upon these challenges unclear. We aimed to identify the affects of elevated temperature and hypercapnia in isolation and in combination on the metabolic and acid-base regulatory processes of a freshwater crayfish, Procambarus clarkii. Crayfish were exposed to freshwater conditions that may be prevalent by the year 2100 and metabolic responses were determined after 14-days of exposure. In addition, changes in branchial mRNA expression of acid-base linked transporters were investigated. Interactions between exposure conditions influenced extracellular pH as well as the nitrogen physiology and routine metabolic rate of the crayfish. Crayfish exposed to individual and combined elevations in temperature and/or hypercapnia maintained an extracellular pH similar to that of control crayfish. Dual-stressor exposed crayfish seem to elevate the importance of ammonium as an excretable acid-equivalent based on an overall increase in the branchial mRNA expression of transporters related to ammonia excretion including the Na+/K+-ATPase, Rhesus-protein, and the V-type H+-ATPase. Overall, hypercapnia and dual-stressor conditions caused a metabolic depression that may have long-lasting consequences such as limited locomotion, growth, and reproduction. Future generations of crayfish given the chance to adapt over several generations may ameliorate these consequences.

Effects of Dietary Shifts on Ontogenetic Development of Metabolic Rates in Age 0 Lake Sturgeon (Acipenser fulvescens).

AbstractIn many fish species, ontogenetic dietary shifts cause changes in both quantitative and qualitative intake of energy, and these transitions can act as significant bottlenecks in survival within a given year class. In the present study, we estimated routine metabolic rate (RMR) and forced maximum metabolic rate (FMR) in age 0 lake sturgeon (Acipenser fulvescens) on a weekly basis from 6 to 76 days posthatch (dph) within the same cohort of fish. We were particularly interested in the period of dietary transition from yolk to exogenous feeding between 6 and 17 dph and as the fish transitioned from an artemia-based diet to a predominantly bloodworm diet between 49 and 67 dph. Measurement of growth rate and energy density throughout indicated that there was a brief period of growth arrest during the transition from artemia to bloodworm. The highest mass-specific RMR (mg O2 kg-1 h-1) recorded throughout the first 76 d of development occurred during the yolk sac phase and during transition from artemia to bloodworm. Similarly, diet transition from artemia to bloodworm-when growth arrest was observed-increased scaled RMR (i.e., mg O2 kg-0.89 h-1), and it did not significantly differ from scaled FMR. Log-log relationships between non-mass-specific RMR or FMR (i.e., mg O2 h-1) and body mass significantly changed as the growing fish adapted to the nutritional differences of their primary diet. We demonstrate that dietary change during early ontogeny has consequences for growth that may reflect altered metabolic performance. Results have implications for understanding cohort and population dynamics during early life and effective management for conservation fish hatcheries.

The effects of population and thermal acclimation on the growth, condition and cold responsive mRNA expression of age-0 lake sturgeon (Acipenser fulvescens).

In Manitoba, Canada, wild lake sturgeon (Acipenser fulvescens) populations exist along a latitudinal gradient and are reared in hatcheries to bolster threatened populations. We reared two populations of lake sturgeon, one from each of the northern and southern ends of Manitoba and examined the effects of typical hatchery temperatures (16°C) as well as 60-day acclimation to elevated rearing temperatures (20°C) on mortality, growth and condition throughout early development. Additionally, we examined the cold shock response, which may be induced during stocking, through the hepatic mRNA expression of genes involved in the response to cold stress and homeoviscous adaptation (HSP70, HSP90a, HSP90b, CIRP and SCD). Sturgeon were sampled after 1 day and 1 week following stocking into temperatures of 8, 6 and 4°C in a controlled laboratory environment. The southern population showed lower condition and higher mortality during early life than the northern population while increased rearing temperature impacted the growth and condition of developing northern sturgeon. During the cold shock, HSP70 and HSP90a mRNA expression increased in all sturgeon treatments as stocking temperature decreased, with higher expression observed in the southern population. Expression of HSP90b, CIRP and SCD increased as stocking temperature decreased in northern sturgeon with early acclimation to 20°C. Correlation analyses indicated the strongest molecular relationships were in the expression of HSP90b, CIRP and SCD, across all treatments, with a correlation between HSP90b and body condition in northern sturgeon with early acclimation to 20°C. Together, these observations highlight the importance of population and rearing environment throughout early development and on later cellular responses induced by cold stocking temperatures.

Effects of temperature and food availability on liver fatty acid composition and plasma cortisol concentration in age-0 lake sturgeon: Support for homeoviscous adaptation.

Overwintering survival in north temperate fishes involves a series of adaptive responses to multiple environmental stressors. Homeoviscous adaptation includes changes in membrane lipid composition in response to reduced environmental temperature, which may be driven by changes in hormones involved in the endocrine stress response. We examined how reduced temperature and food availability may act in concert to influence hepatic fatty acid composition of phospholipids and triglycerides, in addition to plasma concentration of cortisol in age-0 lake sturgeon (A. fulvescens). At 153 days post hatch (dph), temperature was decreased from 16 °C to 1 °C at a rate of 0.5 °C per day, and at 200 dph, fish were either fed every other day or deprived of food for 45 days to simulate an overwintering event. Liver fatty acid composition of phospholipids and triglycerides were assessed before temperature manipulation (16 °C; 153 dph), when fish had been at 1 °C for 16 days (199 dph), 25 days of overwintering (225 dph) and 45 days of overwintering (245 dph). Plasma cortisol concentration was assessed at 153, 225 and 245 dph. When temperature was decreased, both mono- and polyunsaturated fatty acids significantly increased in phospholipids and triglycerides. Total omega-6 fatty acids significantly increased in phospholipids while total omega-3 fatty acids did not. During the simulated overwintering, there was no obvious difference in fatty acids of phospholipids and triglycerides between diet treatments and no difference in circulating cortisol concentration between baseline and post-stressed fish in the fasted group. Our results provide support for homeoviscous adaptation to cold temperatures in lake sturgeon.

Comparison of methods to quantify metabolic rate and its relationship with activity in larval lake sturgeon Acipenser fulvescens.

Until recently most studies have focussed on method development for metabolic rate assessment in adult and/or juvenile fish with less focus on measurement of oxygen consumption (MO2 ) during early life history stages, including fast-growing larval fish and even less focus on nonteleostean species. In the present study we evaluated measurement techniques for standard metabolic rate (SMR), maximum metabolic rate (MMR) and aerobic scope in an Acipenseriform, the lake sturgeon Acipenser fulvescens, throughout the first year of life. Standardized forced exercise protocols to assess MMR were conducted for 5 or 15 min before or after measurement of SMR. We used different levels of oxygen decline during the measurement period of MMR post forced exercise to understand the influence these may have on the calculation of MMR. Opercular rate and tail beat frequencies were recorded by video as measures of behaviours and compared to metabolic rate recorded over a 24 h period. Results indicate that calculated values for aerobic scope were lower in younger fish. Neither exercise sequence nor exercise duration influenced metabolic rate measurements in the younger fish, but exercise duration did affect measurement of MMR in older fish. Finally, there was no strong correlation between metabolic rate and the measured behaviours in the lake sturgeon at either age. Based on the results, we recommend that a minimum of 6 h of acclimation to the respirometry chamber should be given prior to measuring SMR, a chasing protocol to elicit MMR should ideally be performed at the end of experiment, a short chasing time should be avoided to minimize variation and assessment of MMR should balance measurement limitations of the probes along with when and for how long oxygen consumption is measured.

Love thy neighbor: Social buffering following exposure to an acute thermal stressor in a gregarious fish, the lake sturgeon (Acipenser fulvescens).

Social buffering is a phenomenon where the presence of conspecifics reduces an animal's stress response. Well known in mammals, social buffering was recently described in fishes exhibiting pronounced social hierarchies. Lake sturgeon (Acipenser fulvescens) are a gregarious rather than hierarchical fish. Therefore, we tested their capacity for social buffering following exposure to an acute thermal stress. Isolated or grouped (three or six similarly sized conspecifics) age-0 lake sturgeon were exposed to a critical thermal maximum (CTmax) test. We measured the endocrine and cellular response to acute thermal shock by assessing whole body cortisol concentration and mRNA expression of steroidogenic acute regulatory protein (StAR) and heat shock proteins (hsp90a, hsp90b, and hsp70) during recovery from the CTmax test. Isolation or grouping had no effect on CTmax. Whole body cortisol concentrations in isolated fish were approximately three-fold higher than in grouped fish 1 h post-CTmax and two-fold higher than grouped fish 20 h post-CTmax. Similarly, 1 h post-CTmax, mRNA expression of StAR, hsp90a, hsp90b and hsp70 were three to four-fold higher in isolated fish compared to groups of three and six fish. At 20 h post-CTmax, expression of StAR was approximately two-fold higher in isolated fish, but expression of hsp90a, hsp90b, and hsp70 was not significantly different between isolated and grouped fish. While conspecific presence had no effect on CTmax, the significant reduction of endocrine and cellular stress markers post-CTmax in grouped fish strongly suggests that lake sturgeon may use social buffering to combat potential deleterious effects of exposure to heat stress.

Effects of acclimation temperature on the thermal physiology in two geographically distinct populations of lake sturgeon (Acipenser fulvescens).

Temperature is one of the most important abiotic factors regulating development and biological processes in ectotherms. By 2050, climate change may result in temperature increases of 2.1-3.4°C in Manitoba, Canada. Lake sturgeon, Acipenser fulvescens, from both northern and southern populations in Manitoba were acclimated to 16, 20 and 24°C for 30 days, after which critical thermal maximum (CTmax) trials were conducted to investigate their thermal plasticity. We also examined the effects of temperature on morphological and physiological indices. Acclimation temperature significantly influenced the CTmax, body mass, hepatosomatic index, metabolic rate and the mRNA expression of transcripts involved in the cellular response to heat shock and hypoxia (HSP70, HSP90a, HSP90b, HIF-1α) in the gill of lake sturgeon. Population significantly affected the above phenotypes, as well as the mRNA expression of Na+/K+ ATPase-α1 and the hepatic glutathione peroxidase enzyme activity. The southern population had an average CTmax that was 0.71 and 0.45°C higher than the northern population at 20 and 24°C, respectively. Immediately following CTmax trials, mRNA expression of HSP90a and HIF-1α was positively correlated with individual CTmax of lake sturgeon across acclimation treatments and populations (r = 0.7, r = 0.62, respectively; P < 0.0001). Lake sturgeon acclimated to 20 and 24°C had decreased hepatosomatic indices (93 and 244% reduction, respectively; P < 0.0001) and metabolic suppression (27.7 and 42.1% reduction, respectively; P < 0.05) when compared to sturgeon acclimated to 16°C, regardless of population. Glutathione peroxidase activity and mRNA expression Na+/K+ ATPase-α1 were elevated in the northern relative to the southern population. Acclimation to 24°C also induced mortality in both populations when compared to sturgeon acclimated to 16 and 20°C. Thus, increased temperatures have wide-ranging population-specific physiological consequences for lake sturgeon across biological levels of organization.

Influence of a dynamic rearing environment on development of metabolic phenotypes in age-0 Lake Sturgeon, Acipenser fulvescens.

Environment-phenotype interactions are the most pronounced during early life stages and can strongly influence metabolism and ultimately ecological fitness. In the present study, we examined the effect of temperature [ambient river temperature (ART) vs ART+2°C], dissolved oxygen (DO; 100% vs 80%) and substrate (presence vs absence) on standard metabolic rate, forced maximum metabolic rate and metabolic scope with Fulton's condition factor (K), energy density (ED) and critical thermal maximum (CTmax) in age-0 Lake Sturgeon, Acipenser fulvescens, before and after a simulated overwintering event. We found that all the environmental variables strongly influenced survival, K, ED and CTmax. Fish reared in elevated temperature showed higher mortality and reduced K pre-winter at 127 days post-hatch (dph). Interestingly, we did not find any significant difference in terms of metabolic rate between treatments at both sampling points of pre- and post-winter. Long-term exposure to 80% DO reduced ED in Lake Sturgeon post-winter at 272 dph. Our data suggest that substrate should be removed at the onset of exogenous feeding to enhance the survival rate of age-0 Lake Sturgeon in the first year of life. Effects of early rearing environment during larval development on survival over winter are discussed with respect to successful recruitment of stock enhanced Lake Sturgeon, a species that is at risk throughout its natural range.

Impact of climate change on the American lobster (Homarus americanus): Physiological responses to combined exposure of elevated temperature and pCO2.

The physiological consequences of exposing marine organisms to predicted future ocean scenarios, i.e. simultaneous increase in temperature and pCO2, have only recently begun to be investigated. Adult American lobster (Homarus americanus) were exposed to either current (16 °C, 47 Pa pCO2, pH 8.10) or predicted year 2300 (20 °C, 948 Pa pCO2, pH 7.10) ocean parameters for 14-16 days prior to assessing physiological changes in their hemolymph parameters as well as whole animal ammonia excretion and resting metabolic rate. Acclimation of lobster simultaneously to elevated pCO2 and temperature induced a prolonged respiratory acidosis that was only partially compensated for via accumulation of extracellular HCO3- and ammonia. Furthermore, acclimated animals possessed significantly higher ammonia excretion and oxygen consumption rates suggesting that future ocean scenarios may increase basal energetic demands on H. americanus. Enzyme activity related to protein metabolism (glutamine dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) in hepatopancreas and muscle tissue were unaltered in future ocean scenario exposed animals; however, muscular citrate synthase activity was reduced suggesting that, while protein catabolism may be unchanged, the net energetic output of muscle may be compromised in future scenarios. Overall, H. americanus acclimated to ocean conditions predicted for the year 2300 appear to be incapable of fully compensating against climate change-related acid-base challenges and experience an increase in metabolic waste excretion and oxygen consumption. Combining our study with past literature on H. americanus suggests that the whole lifecycle from larvae to adult stages is at risk of severe growth, survival and reproductive consequences due to climate change.