Journal of Experimental Biology

Glucose, which plays a central role in providing energy for metabolism, is primarily stored as glycogen. The synthesis and degradation of glycogen are mainly initialized by glycogen synthase (GS) and glycogen phosphorylase (GP),respectively. The present study aimed to examine the glycogen metabolism in fish liver and gills during acute exposure to seawater. In tilapia(Oreochromis mossambicus) gill, GP, GS and glycogen were immunocytochemically colocalized in a specific group of glycogen-rich (GR)cells, which are adjacent to the gill's main ionocytes, mitochondrion-rich(MR) cells. Na+/K+-ATPase activity in the gills, protein expression and/or activity of GP and GS and the glycogen content of the gills and liver were examined in tilapia after their acute transfer from freshwater(FW) to 25‰ seawater (SW). Gill Na+/K+-ATPase activity rapidly increased immediately after SW transfer. Glycogen content in both the gills and liver were significantly depleted after SW transfer, but the depletion occurred earlier in gills than in the liver. Gill GP activity and protein expression were upregulated 1–3 h post-transfer and eventually recovered to the normal level as determined in the control group. At the same time, GS protein expression was downregulated. Similar changes in liver GP and GS protein expression were also observed but they occurred later at 6–12 h post-transfer. In conclusion, GR cells are initially stimulated to provide prompt energy for neighboring MR cells that trigger ion-secretion mechanisms. Several hours later, the liver begins to degrade its glycogen stores for the subsequent energy supply.

The trout heart is 10-fold more sensitive to Ca2+ than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca2+ affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca2+ affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca2+ activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca2+, its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca2+ affinity was increased ∼1.8-fold. This suggests that trout cTnI contributes to the high Ca2+ sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca2+ affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

The diatom-derived aldehyde 2-trans-4-trans-decadienal(DD) was tested as an apoptogenic inducer in both copepod and sea urchin embryos, using terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL), DNA fragmentation profiling (laddering) and an assay for caspase-3 activity. DD induced TUNEL positivity and DNA laddering, but not caspase-like activation, in copepod embryos spawned by females fed for 10-15 days the diatom diet Thalassiosira rotula Meunier (in vivo),or when newly spawned eggs were exposed for 1 h to 5 μg ml-1 DD(in vitro). To our knowledge, this is the first time that evidence for an apoptotic process in copepods has been obtained by cytochemical (TUNEL)and biochemical (DNA fragmentation) approaches. The absence of caspase-like activity in copepod embryos suggests that caspase-independent programmed cell death occurs in these organisms. In sea urchin embryos, DD induced apoptosis and also activated a caspase-3-like protease. The saturated aldehyde decanal induced apoptosis at higher concentrations and after a longer incubation period than DD, indicating that α,β-unsaturation of the molecule,coupled with the aldehyde group, is responsible for the greater biological activity of DD. Since diatoms are an important food source for marine herbivores such as copepods and sea urchins, these findings may help explain why unsaturated aldehydes often induce reproductive failure, with important ecological consequences at the population level.

Reptiles habitually ingest large meals at infrequent intervals, leading to changes in acid–base status as the net secretion of acid to the stomach causes a metabolic alkalosis (the alkaline tide). In chronically cannulated and undisturbed amphibians and reptiles, the pH changes in arterial blood are, nevertheless, reduced by a concomitant respiratory acidosis (increased caused by a relative hypoventilation). Alligators (Alligator mississippiensis) have been reported to exhibit exceptionally large increases in plasma [HCO3−] following feeding, but these studies were based on blood samples obtained by cardiac puncture, so stress and disturbance may have affected the blood gas levels. Furthermore, crocodilian haemoglobin is characterised by a unique binding of HCO3− that act to reduce blood oxygen-affinity, and it has been proposed that this feature safeguards oxygen offloading by counteracting pH effects on blood oxygen-affinity. Therefore, to study acid–base regulation and the interaction between the alkaline tide and oxygen transport in more detail, we describe the arterial blood gas composition of chronically cannulated and undisturbed alligators before and after voluntary feeding (meal size 7.5±1 % of body mass).

Digestion was associated with an approximately fourfold increase in metabolic rate (from 0.63±0.04 to 2.32±0.24 ml O2 min−1 kg−1) and was accompanied by a small increase in the respiratory gas exchange ratio. The arterial of fasting alligators was 60.3±6.8 mmHg (1 mmHg=0.133 kPa) and reached a maximum of 81.3±2.7 mmHg at 96 h following feeding; there was only a small increase in lactate levels, so the increased metabolic rate seems to be entirely aerobic. Plasma [HCO3−] increased from 24.4±1.1 to 36.9±1.7 mmol l−1 (at 24 h), but since arterial increased from 29.0±1.1 to 36.8±1.3 mmHg, arterial pH remained virtually unaffected (changing from 7.51±0.01 to 7.58±0.01 at 24 h). The changes in plasma [HCO3−] were mirrored by equimolar reductions in plasma [Cl−]. The in vitro blood oxygen-affinity was reduced during the post-prandial period, whereas the estimated in vivo blood oxygen-affinity remained virtually constant. This supports the view that the specific HCO3− effect prevents an increased blood oxygen-affinity during digestion in alligators.

Chronic hypoxic incubation is a common tool used to address the plasticity of morphological and physiological characteristics during vertebrate development. In this study chronic hypoxic incubation of embryonic American alligators resulted in both morphological (mass) and physiological changes. During normoxic incubation embryonic mass, liver mass and heart mass increased throughout the period of study, while yolk mass fell. Chronic hypoxia(10%O2) resulted in a reduced embryonic mass at 80% and 90% of incubation. This reduction in embryonic mass was accompanied by a relative enlargement of the heart at 80% and 90% of incubation, while relative embryonic liver mass was similar to the normoxic group. Normoxic incubated alligators maintained a constant heart rate during the period of study, while mean arterial pressure rose continuously. Both levels of hypoxic incubation(15% and 10%O2) resulted in a lower mean arterial pressure at 90%of incubation, while heart rate was lower in the 10%O2 group only. Acute (5 min) exposure to 10%O2 in the normoxic group resulted in a biphasic response, with a normotensive bradycardia occurring during the period of exposure and a hypertensive tachycardic response occurring during recovery. The embryos incubated under hypoxia also showed a blunted response to acute hypoxic stress. In conclusion, the main responses elicited by chronic hypoxic incubation, namely, cardiac enlargement, blunted hypoxic response and systemic vasodilation, may provide chronically hypoxic embryos with a new physiological repertoire for responding to hypoxia.

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by 3H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10%saturation (0.67±0.01 mg l–1 at 28±1°C). This depression in RoMR was accompanied by a 50–60% decrease in liver,heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

Recent palaeoatmospheric models suggest large-scale fluctuations in ambient oxygen level over the past 550 million years. To better understand how global hypoxia and hyperoxia might have affected the growth and physiology of contemporary vertebrates, we incubated eggs and raised hatchlings of the American alligator. Crocodilians are one of few vertebrate taxa that survived these global changes with distinctly conservative morphology. We maintained animals at 30°C under chronic hypoxia (12% O2), normoxia (21%O2) or hyperoxia (30% O2). At hatching, hypoxic animals were significantly smaller than their normoxic and hyperoxic siblings. Over the course of 3 months, post-hatching growth was fastest under hyperoxia and slowest under hypoxia. Hypoxia, but not hyperoxia, caused distinct scaling of major visceral organs–reduction of liver mass, enlargement of the heart and accelerated growth of lungs. When absorptive and post-absorptive metabolic rates were measured in juvenile alligators, the increase in oxygen consumption rate due to digestion/absorption of food was greatest in hyperoxic alligators and smallest in hypoxic ones. Hyperoxic alligators exhibited the lowest breathing rate and highest oxygen consumption per breath. We suggest that,despite compensatory cardiopulmonary remodelling, growth of hypoxic alligators is constrained by low atmospheric oxygen supply, which may limit their food utilisation capacity. Conversely, the combination of elevated metabolism and low cost of breathing in hyperoxic alligators allows for a greater proportion of metabolised energy to be available for growth. This suggests that growth and metabolic patterns of extinct vertebrates would have been significantly affected by changes in the atmospheric oxygen level.

Chronic exposure to a low incubation temperature clearly slows the development of poikilothemic chicken embryos (or any other poikilotherms), but little is known about the more subtle developmental effects of temperature,especially on physiological regulatory systems. Consequently, two populations of chicken embryos were incubated at 38°C and 35°C. When compared at the same development stage, incubation temperature had no significant impact on embryonic survival or growth. Moreover, the relative timing of major developmental landmarks (e.g. internal pipping), expressed as a percentage of development, was unaffected by temperature. The ability to maintain the rate of oxygen consumption(V̇O2) during an acute drop in ambient temperature (Ta) improved from Hamburger–Hamilton (HH) stages 39–40 to 43–44 in the 38°C but not the 35°C populations. Late stage (HH43–44) embryos incubated at 38°C could maintain V̇O2(approximately 27–33 μl g–1 min–1)during an acute drop in Ta to approximately 30°C. However, at the same stage 35°C embryos acutely measured at 38°C were unable to similarly maintain their V̇O2, which fell as soon as Ta reached 36°C. Thus, while hypothermic incubation does not affect gross development (other than would be predicted from a simple effect of Q10), there is a significant delay in the relative timing of the onset of thermoregulatory ability induced by hypothermic incubation.

Transepithelial Cl− secretion in vertebrates is accomplished by a secondary active transport process brought about by the coordinated activity of apical and basolateral transport proteins. The principal basolateral components are the Na+/K+-ATPase pump, the Na+/K+/2Cl− cotransporter (symporter) and a K+ channel. The rate-limiting apical component is a cyclic-AMP-stimulated Cl− channel. As postulated nearly two decades ago, the net Cl− movement from the blood to the lumen involves entry into the epithelial cells with Na+ and K+, followed by active Na+ extrusion via the pump and passive K+ exit via a channel. Intracellular [Cl−] is raised above electrochemical equilibrium and exits into the lumen when the apical Cl− channel opens. Cl− secretion is accompanied by a passive paracellular flow of Na+. The tubules of the rectal glands of elasmobranchs are highly specialized for secreting concentrated NaCl by this mechanism and hence have served as an excellent experimental model in which to characterize the individual steps by electrophysiological and ion flux measurements. The recent molecular cloning and heterologous expression of the apical Cl− channel and basolateral cotransporter have enabled more detailed analyses of the mechanisms and their regulation. Not surprisingly, since hormones acting through kinases control secretion, both the Cl− channel, which is the shark counterpart of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and the cotransporter are regulated by phosphorylation and dephosphorylation. The primary stimulation of secretion by hormones employing cyclic AMP as second messenger activates CFTR via the direct action of protein kinase A (PKA), which phosphorylates multiple sites on the R domain. In contrast, phosphorylation of the cotransporter by as yet unidentified kinases is apparently secondary to the decrease in intracellular chloride concentration caused by anion exit through CFTR.

Environmental signals can activate neuro-endocrine cascades that regulate various physiological processes. In the present study, we demonstrate that two responses to environmental stress signaling in the crustacean Daphnia magna - hemoglobin accumulation and male offspring production - are co-elevated by the crustacean terpenoid hormone methyl farnesoate and several synthetic analogs. Potency of the hormones with respect to the induction of both hemoglobin and male offspring was highly correlated, suggesting that both processes are regulated by the same terpenoid signaling pathway. Six clones of the D. pulex/pulicaria species complex that were previously characterized as unable to produce male offspring and five clones that were capable of producing males were evaluated for both hemoglobin induction and male offspring production in response to methyl farnesoate. Four of the five male-producing clones produced both hemoglobin and male offspring in response to the hormone. Five of the six non-male-producing clones produced neither hemoglobin nor males in response to the hormone. These results provide additional evidence that both physiological processes are regulated by the same signaling pathway. Furthermore, the results indicate that the non-male-producing clones are largely defective in some methyl farnesoate signaling component, downstream from methyl farnesoate synthesis but upstream from the genes regulated by the hormone. A likely candidate for the site of the defect is the methyl farnesoate receptor. As a consequence of this defect,non-male-producing clones have lost their responsiveness to environmental signals that are transduced by this endocrine pathway. This defect in signaling would be likely to enhance population growth in stable environments due to the elimination of males from the population, assuming that other processes critical to population growth are not also compromised by this defect.