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.

Absence of a functional gut microbiome impairs host amino acid metabolism in the Pacific spiny dogfish (Squalus suckleyi).

Nitrogen recycling and amino acid synthesis are two notable ways in which the gut microbiome can contribute to host metabolism, and these processes are especially important in nitrogen limited animals. Marine elasmobranchs are nitrogen limited as they require substantial amounts of this element to support urea-based osmoregulation. However, following antibiotic-induced depletion of the gut microbiome elasmobranchs are known to experience a significant decline in circulating urea and employ compensatory nitrogen conservation strategies such as reduced urea and ammonia excretion. We hypothesized that the elasmobranch gut microbiome transforms dietary and recycled nutrients into amino acids, supporting host carbon and nitrogen balance. Here, using stable isotope analyses, we found that depleting the gut microbiome of Pacific spiny dogfish (Squalus suckleyi) resulted in a significant reduction to the incorporation of supplemented dietary 15N into plasma amino acids, notably those linked to nitrogen handling and energy metabolism, but had no effect on gut amino acid transport. These results demonstrate the importance of gut microbes to host amino acid pools and the unique nitrogen handling strategy of marine elasmobranchs. More broadly, these results elucidate how the gut microbiome contributes to organismal homeostasis, which is likely a ubiquitous phenomenon across animal populations.

The chondrichthyan glucagon-like peptide 3 regulates hepatic ketone metabolism in the Pacific spiny dogfish Squalus suckleyi.

Chondrichthyans have a novel proglucagon-derived peptide, glucagon-like peptide (GLP)-3, in addition to GLP-1 and GLP-2 that occur in other vertebrates. Given that the GLPs are important regulators of metabolic homeostasis across vertebrates, we sought to investigate whether GLP-3 displays functional actions on metabolism within a representative chondrichthyan, the Pacific spiny dogfish Squalus suckleyi. There were no observed effects of GLP-3 perfusion (10 nM for 15 min) on the rate of glucose or oleic acid acquisition at the level of the spiral valve nor were there any measured effects on intermediary metabolism within this tissue. Despite no effects on apparent glucose transport or glycolysis in the liver, a significant alteration to ketone metabolism occurred. Firstly, ketone flux through the perfused liver switched from a net endogenous production to consumption following hormone application. Accompanying this change, significant increases in mRNA transcript abundance of putative ketone transporters and in the activity of ß-hydroxybutyrate dehydrogenase (a key enzyme regulating ketone flux in the liver) were observed. Overall, while these results show effects on hepatic metabolism, the physiological actions of GLP are distinct between this chondrichthyan and those of GLP-1 on teleost fishes. Whether this is the result of the particular metabolic dependency on ketone bodies in chondrichthyans or a differential function of a novel GLP remains to be fully elucidated.

Understanding olfactory and behavioural responses to dietary cues in age-1 lake sturgeon Acipenser fulvescens.

Detection of environmental cues is essential for all vertebrates and is typically established by the olfactory epithelium and olfactory sensory neurons (OSNs). In fishes, microvillous and ciliated OSNs are the principal types, typically detecting amino acids and bile salts, respectively. Activation of OSN receptors by specific ligands initiate downstream signal processing often leading to behavioural responses. In this study we used electrophysiological and behavioural techniques to evaluate olfactory detection and behaviour in juvenile lake sturgeon Acipenser fulvescens in response to hatchery- and natural dietary cues. We hypothesized that electro-olfactogram (EOG) and behavioural responses would be dependent on diet type. We predicted that inhibition of the phospholipase C/inositol 1,4,5-triphosphate (PLC/IP3) secondary transduction pathway would reduce EOG responses to dietary cues and, inhibition of the adenylyl cyclase/adenosine 3,5-cyclic monophosphate (cAMP) pathway, would have no effect. Furthermore, we predicted a strong EOG response would be manifested in a change in behaviour. We observed that both the PLC/IP3 and cAMP pathways were significantly involved in the detection of dietary cues. However, EOG responses did not manifest to behavioural responses, although the foraging activity to the hatchery cue was significantly greater compared to the control. Our results support the notion that lake sturgeon raised in a hatchery and fed a commercial pelleted diet may become accustomed to it prior to release into the wild. Further, this study suggests that, in conservation aquaculture settings, lake sturgeon should be exposed to natural dietary cues prior to release as one strategy to promote food recognition.

In situ method for the determination of nutrient acquisition and its hormonal regulation in the spiral valve of two chondrichthyan fishes.

Chondrichthyans play an important role in nutrient cycling of many marine ecosystems, yet little is known about their nutritional physiology particularly relating to nutrient acquisition in the spiral valve intestine. This unique organ poses challenges for examining nutrient transport physiology using traditional reductionist methods owing to its scroll-like morphology. Thus, we established a method for the characterization of nutrient uptake rates in two representative chondrichthyans, the Pacific spiny dogfish (Squalus suckleyi) and the Pacific spotted ratfish (Hydrolagus colliei). We validated a dual-cannulation method wherein perfusate was circulated through the vasculature of the spiral valve via the anterior and posterior intestinal arteries, and [14C]glucose or [3H]oleic acid was accumulated from the static spiral valve lumen into the anterior and posterior intestinal veins. Radiotracer accumulated at a stable rate in the venous effluent in comparison with measures of mucosal disappearance. Interestingly, similar anterior and posterior glucose uptake was observed in dogfish, yet significantly more oleic acid was accumulated in the posterior veins of ratfish. Further validation of the preparation in dogfish demonstrated sodium dependence of glucose transport as well as an effect of bovine insulin administration to the arterial circulation. Each of these manipulations resulted in significant differences in glucose handling between the anterior and posterior veins, suggestive of heretofore unknown heterogenous functions along the intestine. This preparation demonstrates a new and reliable method for the measurement of nutrient acquisition and regulation thereof in a unique digestive organ. Furthermore, it presents avenues for investigation of differential functional along the spiral valve.NEW & NOTEWORTHY We describe a novel dual cannulation method for investigating radiolabeled nutrient uptake from a unique organ, the spiral valve. Furthermore, we identify functional differences in nutrient transport along the length of the spiral valve which consists of a homogenous gross morphology. Finally, this method reveals a useful way in which to manipulate the arterial supply to better understand postprandial physiology as it varies with metabolites and endocrine factors.

An adapted liver perfusion in a shark species, Squalus suckleyi: investigation of energy mobilization.

The liver is an essential energy storage organ in vertebrates. In teleosts and elasmobranchs, previous studies examining hepatic energy balance have used isolated hepatocytes. Although these studies have been informative, the high-fat content in the elasmobranch liver limits isolation of hepatocytes and therefore the utility of this method to understand hepatic metabolic processes. In the present study, we developed an in situ liver perfusion in the North Pacific spiny dogfish Squalus suckleyi. Perfusions were conducted by cannulating the hepatic portal vein (inflowing cannulation) and the sinus venosus through the heart (outflowing cannulation). Changes in major elasmobranch metabolites (glucose and 3-hydroxybutarate [3-HB]) were determined by the arterial (inflow)-venous (outflow) difference in metabolite concentration. Liver preparations were considered viable due to consistent oxygen consumption over 3 h and the maintenance of predictable vasoconstriction following administration of homologous 10-7 M angiotensin II (ANG II). Removal and reintroduction from the perfusate of metabolites showed endogenous 3-HB production in the isolated perfused livers but did not affect glucose balance. However, the arterial-venous difference of both metabolites did not change following perfusion with heterologous insulin and homologous glucagon, which may be due to the glucose intolerant nature of elasmobranchs. Ultimately, we show the viability of this perfusion for the investigation of hepatic energy mobilization in sharks.NEW & NOTEWORTHY We describe a viable liver perfusion in a shark species for the first time as determined by oxygen consumption and hormone-mediated changes in hemodynamics (angiotensin II, ANG II). In addition, removal of major energy metabolites confirms hepatic ketone [3-hydroxybutyrate (3-HB)] production by an elasmobranch liver. Perfusion with heterologous insulin and homologous glucagon did not cause changes in glucose balance, however, possibly demonstrating differences in glucose metabolism in this taxon as compared with more derived vertebrates.

Elevated temperatures reduce population-specific transcriptional plasticity in developing lake sturgeon (Acipenser fulvescens).

Rising mean and variance in temperatures elevates threats to endangered freshwater species such as lake sturgeon, Acipenser fulvescens. Previous research demonstrated that higher temperatures during development result in physiological consequences for lake sturgeon populations throughout Manitoba, Canada, with alteration of metabolic rate, thermal tolerance, transcriptional responses, growth and mortality. We acclimated lake sturgeon (30-60 days post fertilization, a period of high mortality) from northern and southern populations (56°02'46.5″N, 96°54'18.6″W and 50°17'52″N, 95°32'51″W, respectively, separated by approximately 650 km) within Manitoba to current (summer highs of 20-23°C) and future projected (+2-3°C) environmental temperatures of 16, 20 and 24°C for 30 days, and we measured gill transcriptional responses using RNAseq. Transcripts revealed SNPs consistent with genetically distinct populations and transcriptional responses altered by acclimation temperature. There were a higher number of differentially expressed transcripts observed in the southern, compared to the northern, population as temperatures increased, indicating enhanced transcriptional plasticity. Both lake sturgeon populations responded to elevated acclimation temperatures by downregulating the transcription of genes involved in protein synthesis and energy production. Furthermore, there were population-specific thresholds for the downregulation of processes promoting transcriptional plasticity as well as mitochondrial function as the northern population showed decreases at 20°C, while this capacity was not diminished until 24°C in the southern population. These transcriptional responses highlight the molecular impacts of increasing temperatures for divergent lake sturgeon populations during vulnerable developmental periods and the critical influence of transcriptome plasticity on acclimation capacity.

Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

Physiological responses to acute warming at the agitation temperature in a temperate shark.

Thermal tolerance and associated mechanisms are often tested via the critical thermal maximum (CTmax). The agitation temperature is a recently described thermal limit in fishes that has received little mechanistic evaluation. The present study used a temperate elasmobranch fish to test the hypothesis that this thermal tolerance trait is partially set by the onset of declining cardiorespiratory performance and the cellular stress response. Pacific spiny dogfish (Squalus suckleyi) were screened for cardiorespiratory and whole-organism thermal limits to test for associations between thermal performance and tolerance. Then, biochemical markers of secondary stress, aerobic and anaerobic enzyme activities, and molecular markers of cellular stress were determined for various tissues at the agitation temperature and secondary stress markers were determined at CTmax. In dogfish, the agitation temperature was characterised by increased turning activity within experimental chambers and was equal to the temperature at which dogfish exhibited maximum heart rate. Citrate synthase activity increased at the agitation temperature in white muscle relative to unmanipulated dogfish. Furthermore, lactate dehydrogenase activity and accumulated lactate in the plasma and muscle were not affected by acute warming. Cellular stress was apparent in hypothalamus, gill filament and ventricle, denoted by elevated transcript abundance of the stress response gene hsp70 but not the oxygen homeostasis gene hif1α. Conversely, CTmax was characterised by metabolic acidosis driven by anaerobic lactate production, signifying an increased reliance on anaerobic metabolism between the agitation temperature and CTmax. Together, these data provide partial support for our hypothesis, in that cellular stress, but not declining thermal performance, occurred at the agitation temperature.

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.

Nitrogen transporters along the intestinal spiral valve of cloudy catshark (Scyliorhinus torazame): Rhp2, Rhbg, UT.

As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark.

The gut microbiome may influence post-prandial nitrogen handling in an elasmobranch, the Pacific spiny dogfish (Squalus suckleyi).

Nitrogen recycling through the gut microbiome is an important mechanism used throughout vertebrates to reclaim valuable nitrogen trapped in urea. Evidence suggests it may be especially important in nitrogen limited animals, yet little is known about its role in marine elasmobranchs, which are said to be severely nitrogen limited. In the present study we used antibiotics to deplete the gut microbiome of Pacific spiny dogfish and assessed the role of the microbiome in nitrogen handling in both fed and fasted states. In fed animals, antibiotic treatment eliminated the activity of the microbial enzyme urease and reduced cellulase activity by 78%. This reduction in microbial enzyme activity resulted in significantly lower plasma urea levels which then trended upward as urea excretion rates decreased. Ammonia excretion rates were also significantly lower in antibiotic treated fish compared to the control fed. Finally, antibiotic treated fed individuals lost an average of 7.4% of their body mass while the fed controls lost only 1.8% of their body mass. Nitrogen handling in fasted animals was not significantly impacted by a reduction in microbial activity. These results suggest that compromising the gut microbiome significantly influences post-prandial nitrogen handling in spiny dogfish, and that the recycling of urea­nitrogen may be vital to maintaining nitrogen balance in these fish.

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.

Sustained endocrine and exocrine function in the pancreas of the Pacific spiny dogfish post-feeding.

Secretions of the exocrine pancreas contain digestive enzymes integral to the digestive process. The Pacific spiny dogfish (Squalus suckleyi) has a discrete pancreas, divided into two lobes termed the dorsal and ventral lobes. These lobes drain into the anterior intestine via a common duct to enable digestion. Previous studies have identified that the exocrine pancreas produces (co)lipases, chymotrypsin, carboxypeptidase, and low levels of chitinases; however, investigations into other digestive enzymes are limited. We detect the presence of lipase, trypsin, and carbohydrase and show that activities are equivalent between both lobes of the pancreas. Additionally, we sought to investigate the influence of a single feeding event (2% body weight ration of herring by gavage) on enzyme activities over an extended time course (0, 20, 48, 72, 168 h) post-feeding. The results indicate that there are no differences in pancreatic tissue digestive enzyme activities between fed or fasted states. Analysis of acinar cell circumference post-feeding demonstrates a significant increase at 20 and 48 h, that returns to fasting levels by 72 h. No significant changes were observed regarding whole-tissue insulin or glucagon mRNA abundance or with glucose transporter (glut) 1 or 3. Yet, a significant and transient decrease in glut4 and sodium glucose-linked transporter mRNA abundance was found at 48 h post-feeding. We propose that the constant enzyme activity across this relatively large organ, in combination with a relatively slow rate of digestion leads to an evenly distributed, sustained release of digestive enzymes regardless of digestive state.

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.

Ancient fishes and the functional evolution of the corticosteroid stress response in vertebrates.

The neuroendocrine mechanism underlying stress responses in vertebrates is hypothesized to be highly conserved and evolutionarily ancient. Indeed, elements of this mechanism, from the brain to steroidogenic tissue, are present in all vertebrate groups; yet, evidence of the function and even identity of some elements of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is equivocal among the most basal vertebrates. The purpose of this review is to discuss the functional evolution of the HPA/I axis in vertebrates with a focus on our understanding of this neuroendocrine mechanism in the most ancient vertebrates: the agnathan (i.e., hagfish and lamprey) and chondrichthyan fishes (i.e., sharks, rays, and chimeras). A review of the current literature presents evidence of a conserved HPA/I axis in jawed vertebrates (i.e., gnathostomes); yet, available data in jawless (i.e., agnathan) and chondrichthyan fishes are limited. Neuroendocrine regulation of corticosteroidogenesis in agnathans and chondrichthyans appears to function through similar pathways as in bony fishes and tetrapods; however, key elements have yet to be identified and the involvement of melanotropins and gonadotropin-releasing hormone in the stress axis in these ancient fishes warrants further investigation. Further, the identities of physiological glucocorticoids are uncertain in hagfishes, chondrichthyans, and even coelacanths. Resolving these and other knowledge gaps in the stress response of ancient fishes will be significant for advancing knowledge of the evolutionary origins of the vertebrate stress response.

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.

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.

Measurement of 1α hydroxycorticosterone in the Japanese banded houndshark, Triakis scyllium, following exposure to a series of stressors.

An endocrine glucocorticoid response following exposure to a stressor has been well described for many vertebrates. However, despite demonstration of secondary stress responses in a number of elasmobranchs, our understanding of the endocrine control of these responses is lacking. This is largely due to the unusual structure of the dominant corticosteroid in elasmobranch fish, 1α-hydroxycorticosterone (1α-OH-B). Here we describe plasma extraction and HPLC separation procedures that allowed for the measurement of 1α-OH-B and corticosterone from plasma samples in the cannulated, conscious free-swimming Japanese banded houndshark, Triakis scyllium. While patterns of concentration in the plasma for 1α-OH-B and corticosterone were found to be similar in all experiments conducted, circulating levels of 1α-OH-B were consistently 100-fold greater than circulating levels of corticosterone. Immediately following cannulation surgery, circulating levels of 1α-OH-B increased 7-fold compared to pre-surgery levels, while the levels were 11-fold higher than pre-stress levels 30 min post a repeated handling/air-exposure stress. A three week period of fasting resulted in a 22-fold increase in circulating levels of 1α-OH-B in the banded houndshark. This is the first report of direct measurement of changes in circulating levels of the primary corticosteroid in elasmobranch fish, 1α-OH-B, following exposure to a stressor such as handling/air-exposure. Data indicate the steroid may respond similarly to the classic glucocorticoid response, such as cortisol in teleosts.

The effects of digesting a urea-rich meal on North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are nitrogen-limited owing to the requirement of nitrogen for both somatic growth and urea-based osmoregulation, and due to the loss of urea across the gills and kidney as nitrogenous waste. In this study we used in vitro stomach and intestinal gut sacs to investigate the effects of consuming a urea-rich meal (700 mM within a 2% body-mass ration of food-slurry) on nitrogen movement across the gastrointestinal (GI) tract of North Pacific spiny dogfish (Squalus acanthias suckleyi). Plasma urea concentrations did not differ between fasted (359 ± 19 mM), urea-poor fed (340 ± 16 mM), and urea-rich fed (332 ± 24 mM) dogfish. Interestingly, in vitro gut sacs of urea-rich fed dogfish showed no net urea absorption from the lumen over 3 h incubation, which contrasts previously published data on urea-poor fed dogfish that absorb urea from the lumen. In addition, ammonium (NH4+) concentration within the gut sac intestinal lumen significantly increased from 0.62 to 4.35 mM over 3 h. This is likely due to a combination of tissue production and microbial urease activity in the intestine. The overall results highlight the ability of S. a. suckleyi to regulate and maintain internal nitrogen concentrations despite the addition of excess dietary urea.