Differential gene expression indicates modulated responses to chronic and intermittent hypoxia in corallivorous fireworms (Hermodice carunculata).

Climate models predict an increase in extent, frequency, and duration of marine hypoxia events in the twenty first century. A better understanding of organismal responses to hypoxia in individual species is a crucial step for predicting ecosystem responses. We experimentally subjected a common invertebrate, the bearded fireworm (Hermodice carunculata) to two levels of chronic hypoxia and, in a separate experiment, to intermittent hypoxia. We found components of the conserved hypoxia-inducible factor (HIF) pathway and show a modulated response to hypoxia depending on the severity of hypoxic stress: under mild hypoxia, only the HIF-1α subunit is upregulated, while expression of the other subunit, aryl hydrocarbon nuclear translator, only increases significantly at more severe hypoxia levels. The chronic trials revealed down-regulation of genes related to cell adhesion, transport, development and heme-binding, and up-regulation of genes related to glycolysis, oxygen binding, cell differentiation, digestive and reproductive function. The intermittent hypoxia trials revealed an upregulation of heme transporter activity during hypoxia, and our time series analysis characterized nine clusters of genes with similar expression patterns. Our findings suggest that H. carunculata is likely to tolerate, and be resilient to, predicted future hypoxia conditions.

Effects of chronic exposure to 12‰ saltwater on the endocrine physiology of juvenile American alligator (Alligator mississippiensis).

American alligator (Alligator mississippiensis) habitats are prone to saltwater intrusion following major storms, hurricanes or droughts. Anthropogenic impacts affecting hydrology of freshwater systems may exacerbate saltwater intrusion into freshwater habitats. The endocrine system of alligators is susceptible to changes in the environment but it is currently not known how the crocodilian physiological system responds to environmental stressors such as salinity. Juvenile alligators were exposed to 12‰ saltwater for 5 weeks to determine the effects of chronic exposure to saline environments. Following 5 weeks, plasma levels of hormones [e.g. progesterone, testosterone, estradiol, corticosterone, aldosterone (ALDO), angiotensin II (ANG II)] were quantified using liquid chromatography and tandem mass spectrometry. Compared with freshwater-kept subjects, saltwater-exposed alligators had significantly elevated plasma levels of corticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone, testosterone, 17ß-estradiol, estrone and estriol whereas pregnenolone and ANG II were significantly depressed and ALDO levels were unchanged (slightly depressed). On the one hand, saltwater exposure did not affect gene expression of renal mineralocorticoid and glucorticoid and angiotensin type 1 (AT-1) receptors or morphology of lingual glands. On the other hand, saltwater exposure significantly reduced plasma glucose concentrations whereas parameters diagnostic of perturbed liver function (aspartate aminotransferase and alanine aminotransferase) and kidney function (creatinine and creatine kinase) were significantly elevated. Except for plasma potassium levels (K+), plasma ions Na+ and Cl- were significantly elevated in saltwater alligators. Overall, this study demonstrated significant endocrine and physiological effects in juvenile alligators chronically exposed to a saline environment. Results provide novel insights into the effects of a natural environmental stressor (salinity) on the renin-angiotensin-aldosterone system and steroidogenesis of alligators.

Temperature and species-specific effects on ß3-adrenergic receptor cardiac regulation in two freshwater teleosts: Channel catfish (Ictalurus punctatus) and common carp (Cyprinus carpio).

ß3-adrenergic receptors (AR) are important in teleost cardiovascular regulation. To date, it is unknown whether temperature acclimation changes ß3-AR functionality and consequently the involvement of this AR subtype in teleost cardiac regulation. Common carp (Cyprinus carpio) were acclimated at 12 °C or 23 °C (minimum 3 weeks) after which cardiovascular variables (cardiac output (Q), stroke volume (Sv) and heart rate (fH)) were measured upon injection of the ß3-AR agonist, BRL(37344), and antagonist, SR(59230A). In both 12 °C and 23 °C acclimated carp, BRL(37344) induced significant increases in fH and Q whereas Sv was significantly decreased. While temperature did not affect the change (increase vs. decrease) in cardiac variables, the magnitude and on-set of responses differed. For instance, fH, Sv and Q responded significantly faster to ß3-AR stimulation in 23 °C carp. In contrast, maximum responses of fH and Q were significantly higher in 23 °C carp whereas the maximum response of Sv was significantly greater in 12 °C carp. These findings suggest that temperature acclimation induced changes in ß3-AR receptor functionality (e.g. density and/or affinity). Stimulation of ß3-ARs in 23 °C acclimated channel catfish (Ictalurus punctatus) caused significant increases in fH, Sv and Q. The increase in Sv was opposite to the decrease observed in 23 °C acclimated common carp. SR(59230A) induced significant decreases in Sv and Q but had no effect in carp (23 °C). Results suggest species diversity in the density and affinity or structure of ß3-ARs which may explain the different cardiac responses to ß3-AR ligands.

The interactive effects of a gradual temperature decrease and long-term food deprivation on cardiac and hepatic blood flows in rainbow trout (Oncorhynchus mykiss).

The aim of the present study was to determine the extent to which the fish liver is perfused with blood. Transonic® flow probes were therefore implanted around the ventral aorta and hepatic vein(s) to record baseline blood flows in rainbow trout (Oncorhynchus mykiss) previously held under two different feeding regimes (food-deprived or fed to satiation, 8-12 weeks). Fish from both groups were exposed to a gradual temperature decrease (12°C to 5°C) and physical disturbance. Cardiac output (Q), stroke volume (Sv) and hepatic venous blood flow (HVBF) were significantly reduced in food-deprived trout at 12°C. Heart rate was not significantly affected by nutritional status, but was significantly reduced when temperature was decreased to 5°C. Physically disturbing each fish at 12°C and 5°C showed that the performance capacity of the heart was not affected by food deprivation as the capacity to increase Q and Sv was not reduced in the food-deprived group. Overall this study showed that food deprivation in rainbow trout reduced cardiac and hepatic blood flows. However, long-term food deprivation did not affect the capacity of the heart to acutely increase performance.

Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS.

Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC-MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20ß-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters™ Sunfire C(18) column (2.1 mm×50 mm with a 3.5 µm particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17α-hydroxypregnenolone, progesterone and17α,20ß-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of ≥3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20ß-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.

Cod (Gadus morhua) cardiorespiratory physiology and hypoxia tolerance following acclimation to low-oxygen conditions.

Previous research has shown that hypoxia-acclimated Atlantic cod (Gadus morhua) have significantly reduced cardiac function but can consume more oxygen for a given cardiac output (Q). However, it is not known (1) which physiological changes permit a greater "oxygen pulse" (oxygen consumed per mL of blood pumped) in hypoxia-acclimated individuals or (2) whether chronic exposure to low-oxygen conditions improves the hypoxia tolerance of cod. Thus, we exposed normoxia- and hypoxia-acclimated (> 6 wk at a water oxygen partial pressure [P(w)O(2)] ~8-9 kPa) cod to a graded normoxia challenge until loss of equilibrium occurred while recording the following cardiorespiratory variables: oxygen consumption (MO(2)), ventilatory rate, cardiac function (Q, heart rate f(H), and stroke volume S(V)), ventral aortic blood pressure (P(VA)), venous oxygen partial pressure (P(v)O(2)) and oxygen content (C(v)O(2)), plasma catecholamines, and blood hemoglobin ([Hb]) and hematocrit (Hct). In addition, we performed in vitro hemoglobin oxygen binding curves to examine whether hypoxia acclimation influences hemoglobin functional properties. Numerous physiological adjustments occurred in vivo during the > 6 wk of hypoxia acclimation: that is, increased f(H), decreased S(V) and Q, elevated [Hb], enhanced tissue oxygen extraction (by 10% at a P(w)O(2) of 20 kPa), and a more robust stress response as evidenced by circulating catecholamine levels that were two to eight times higher when fish were acutely exposed to severe hypoxia. In contrast, chronic hypoxia had no significant effect on the affinity of hemoglobin for oxygen, on in vitro hemoglobin oxygen carrying capacity, or on the cod's hypoxia tolerance (H(crit); the P(w)O(2) at which the fish lost equilibrium, which was 4.3 ± 0.2 and 4.8 ± 0.3 kPa in normoxia- and hypoxia-acclimated fish, respectively). These data suggest that while chronic hypoxia results in numerous physiological adjustments, these changes do not improve the cod's capacity to tolerate low-oxygen conditions.

In situ cardiac function in Atlantic cod (Gadus morhua): effects of acute and chronic hypoxia.

Recent in vivo experiments on Atlantic cod (Gadus morhua) acclimated to chronic hypoxia (6-12 weeks at 10 degrees C; Pw(O(2)) approximately 8-9 kPa) revealed a considerable decrease in the pumping capacity of the heart. To examine whether this diminished cardiac performance was due to the direct effects of chronic moderate hypoxia on the myocardium (as opposed to alterations in neural and/or hormonal control), we measured the resting and maximum in situ function of hearts from normoxia- and hypoxia-acclimated cod: (1) when initially perfused with oxygenated saline; (2) at the end of a 15 min exposure to severe hypoxia (P(O(2)) approximately 0.6 kPa); and (3) 30 min after the hearts had been reperfused with oxygenated saline. Acclimation to hypoxia did not influence resting (basal) in situ cardiac performance during oxygenated or hypoxic conditions. However, it caused a decrease in maximum cardiac output (Q(max)) under oxygenated conditions (from 49.5 to 40.3 ml min(-1) kg(-1); by 19%), that was due to diminished values for maximum stroke volume (V(S)) and scope for V(S). Severe hypoxia reduced in both groups to approximately 20 ml min(-1) kg(-1), yet, the hearts of hypoxia-acclimated fish were better able to sustain this level of Q under hypoxia, and the recovery of Q(max) (as compared with initial values under oxygenated conditions) was significantly improved (94% vs 83%). These data show that acclimation to hypoxia has a direct effect on cod myocardial function and/or physiology, and suggest that the cod heart shows some adaptations to prolonged hypoxia.

Effect of acute and chronic hypoxia on the swimming performance, metabolic capacity and cardiac function of Atlantic cod (Gadus morhua).

Low water oxygen content (hypoxia) is a common feature of many freshwater and marine environments. However, we have a poor understanding of the degree to which diminished cardiac function contributes to the reduction in fish swimming performance concomitant with acute exposure to hypoxia, or how fish cardiorespiratory physiology is altered by, or adapts to, chronic hypoxia. Thus, we acclimated adult Atlantic cod (Gadus morhua) to either approximately 8-9 kPa O(2) (40-45% air saturation) or approximately 21 kPa O(2) (100% air saturation; normoxia) for 6-12 weeks at 10 degrees C, and subsequently measured metabolic variables [routine oxygen consumption (M(O(2)), maximum (M(O(2)), metabolic scope] and cardiac function (cardiac output, Q; heart rate, f(H); and stroke volume, V(S)) in these fish during critical swimming speed (U(crit)) tests performed at both levels of water oxygenation. Although surgery (flow probe implantation) reduced the U(crit) of normoxia-acclimated cod by 14% (from 1.74 to 1.50 BL s(-1)) under normoxic conditions, exposure to acute hypoxia lowered the U(crit) of both groups (surgery and non-surgery) by approximately 30% (to 1.23 and 1.02 BL s(-1), respectively). This reduction in swimming performance was associated with large decreases in maximum M(O(2)) and metabolic scope (> or = 50%), and maximum f(H) and Q (by 16 and 22%), but not V(S). Long-term acclimation to hypoxia resulted in a significant elevation in normoxic metabolic rate as compared with normoxia-acclimated fish (by 27%), but did not influence normoxic or hypoxic values for U(crit), maximum M(O(2)) or metabolic scope. This was surprising given that resting and maximum values for Q were significantly lower in hypoxia-acclimated cod at both levels of oxygenation, because of lower values for V(S). However, hypoxia-acclimated cod were able to consume more oxygen for a given cardiac output. These results provide important insights into how fish cardiorespiratory physiology is impacted by short-term and prolonged exposure to hypoxia, and further highlight the tremendous capacity of the fish cardiorespiratory system to deal with environmental challenges.

Expression levels of genes associated with oxygen utilization, glucose transport and glucose phosphorylation in hypoxia exposed Atlantic cod (Gadus morhua).

Expression level of genes associated with oxygen [cytochrome oxidase 1 (COX1) and myoglobin (Mb)] and glucose utilization [glucose transporters (GLUTs) and hexokinases (HKs)] along with metabolic indices were determined in Atlantic cod (Gadus morhua) subjected to an hypoxic challenge of <45% oxygen saturation for 24 days. There were two closely related HKs considered to be homologues of mammalian HKIs. HKIa and HKIb share 86% sequence identity and are both ubiquitously expressed. Mb was also expressed in many tissues with highest levels occurring in heart. Over the first 15 days of hypoxia there were transient increases in plasma lactate in hypoxic relative to normoxic fish associated with a significant decrease in liver glycogen. Over days 1-6, there were in ten of eleven cases, increased average (with a number of conditions being statistically significant) expression levels of GLUTs (1, 2, 4) and HKs (1a and b) in gill, heart, liver, and white muscle in hypoxic relative to normoxic fish. There were significant increases in COX1 and Mb expression levels in gill by day 24 but no changes in these aerobic indicators in heart or liver. Overall the data suggest a transient increase in genes associated with glucose utilization during the early part of the hypoxic challenge followed by alterations in gene expression in gill.

Cardiovascular and haematological responses of Atlantic cod (Gadus morhua) to acute temperature increase.

For fish to survive large acute temperature increases (i.e. >10.0 degrees C) that may bring them close to their critical thermal maximum (CTM), oxygen uptake at the gills and distribution by the cardiovascular system must increase to match tissue oxygen demand. To examine the effects of an acute temperature increase ( approximately 1.7 degrees C h(-1) to CTM) on the cardiorespiratory physiology of Atlantic cod, we (1) carried out respirometry on 10.0 degrees C acclimated fish, while simultaneously measuring in vivo cardiac parameters using Transonic probes, and (2) constructed in vitro oxygen binding curves on whole blood from 7.0 degrees C acclimated cod at a range of temperatures. Both cardiac output (Q) and heart rate (fh) increased until near the fish's CTM (22.2+/-0.2 degrees C), and then declined rapidly. Q(10) values for Q and fh were 2.48 and 2.12, respectively, and increases in both parameters were tightly correlated with O(2) consumption. The haemoglobin (Hb)-oxygen binding curve at 24.0 degrees C showed pronounced downward and rightward shifts compared to 20.0 degrees C and 7.0 degrees C, indicating that both binding capacity and affinity decreased. Further, Hb levels were lower at 24.0 degrees C than at 20.0 degrees C and 7.0 degrees C. This was likely to be due to cell swelling, as electrophoresis of Hb samples did not suggest protein denaturation, and at 24.0 degrees C Hb samples showed peak absorbance at the expected wavelength (540 nm). Our results show that cardiac function is unlikely to limit metabolic rate in Atlantic cod from Newfoundland until close to their CTM, and we suggest that decreased blood oxygen binding capacity may contribute to the plateau in oxygen consumption.

Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar.

In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10 degrees C, for approximately 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6x faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (M(O(2))), respectively. However, there was no concurrent increase in their maximum M(O(2)), which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5-10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7x higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.