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Cysteine protease activities have been characterized with benzyloxycarbonyl-lysinep-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimpPenaeus vannamei. An optimum pH of 5.1 has been measured. To characterize these cysteine proteases, a hepatopancreas cDNA library was screened by hybridization to a Norway lobster cysteine protease cDNA fragment. Two cDNAs encodingP. vannamei cysteine protease precursors have been cloned and sequenced. The encoded polypeptides have 326 and 322 amino acid residues, respectively, each consisting of partial signal sequences (15 and 10 residues), a pro-region (93 and 94 residues), and a mature enzyme polypeptide (218 residues). Cys25, His159 and Asn175 form the catalytic triad in the putative active site of the mature enzymes. Compared with invertebrate cysteine proteases (Homarus andFasciola), each of the two shrimp enzymes shows 70 and 52% amino acid sequence identity, respectively; 63% identity is shown with rat cathepsin L. Northern hybridization analysis showed the same size for the different cysteine protease transcripts in hepatopancreas tissue (approximately 1.1 kb). During intermolt cycles, variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA.

The codGadus morhua has a closed, compliant swimbladder which occupies 5% of its volume. Pressure changes caused by vertical movements lead to the expansion and compression of the swimbladder gas, and the fish responds to the accompanying changes in density with compensatory swimming movements and the resorption or secretion of gas. A simple physiological model, based on estimates of cardiac output, the blood supply to the swimbladder and the oxygen-carrying capacity of the blood, is developed to set limits to these processes. An apparatus is described for making observations on the rate of buoyancy adaptation by cod subjected to pressure changes within the range 1.15 to 7.50 ATA at temperatures from 0 to 17°C. One hundred and twenty eight experiments were made with 38 cod ranging in length from 25 to 50 cm and in weight from 138 to 1440 g. The results showed that the rate of gas resorption increased markedly with the pressure to which the fish were adapted (from 0.14 ml kg−1 min−1 at 1.5 ATA to 1.54 ml kg−1 min−1 at 7.0 ATA), but not significantly with the weight of the fish or with temperature. In contrast, the rate of gas secretion increased markedly with temperature (from 0.02 ml kg−1 min−1 at 0°C to 0.11 ml kg−1 min−1 at 17°C), increased slightly with pressure, and decreased with the weight of the fish. The rate of gas resorption was much faster than that of gas secretion, and the difference between the two rates increased with pressure. The difference in performance is discussed in relation to the restriction that the swimbladder might impose to the speed and extent of vertical movements. It is suggested that when a closed swimbladder has a hydrostatic function it may be advantageous if neutral buoyancy is maintained only at the upper limit to the diurnal vertical range.

Recent catastrophic global amphibian declines have been partially linked to increases in UV-B radiation as a consequence of stratospheric ozone depletion. Previous studies have shown that in the presence of other environmental stressors including aquatic pH and temperature and the presence of contaminants or pathogens, the lethal effects of UV-B on amphibian larvae are enhanced due to interactions between the stressors. Little is known about the interactions between UV-B and aquatic hypoxia, a common and significant natural stressor of amphibian larvae. We examined the potential effects of UV-B and aquatic hypoxia in combination on embryonic survival, developmental rate, body mass and locomotor performance of embryos and larvae of the striped marsh frog, Limnodynastes peronii. We found that while both UV-B and hypoxia independently had substantial negative effects on the developing embryos of L. peronii, they did not interact in a multiplicative or antagonistic manner. The effects of the stressors in combination were as might be predicted based on the knowledge of their independent actions alone (i.e. an additive effect). In all cases developing embryos exposed to both UV-B and hypoxia were more severely affected than those exposed to either UV-B or hypoxia alone. The results of this study show the importance of examining both the direct actions of individual stressors and how these may be influenced by the presence of other environmental factors.

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69–79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea. Decreased hydration was accompanied by a marked reduction in the resting rate of oxygen consumption, which was inversely correlated with plasma osmolality and urea concentration. In a separate experiment, resting rates of oxygen consumption in fully hydrated frogs receiving injections of saline or saline containing urea did not differ initially; however, upon dehydration, metabolic rates decreased sooner in the urea-loaded frogs than in control frogs. Our findings suggest an important role for urea, acting in concert with dehydration, in the metabolic regulation and energy conservation of hibernating R. sylvatica.

Aquatic hypercarbia, either naturally occurring or anthropogenically induced, can have extensive impacts on aquatic environments and resident organisms. While the impact of acute hypercarbia exposure on the behavior and physiology of fishes has been well studied, relatively little work has examined the physiological impact and acclimation capacity of fishes to chronic hypercarbia. To better understand the impacts of prolonged hypercarbia exposure, largemouth bass were held at ambient CO2 (13 mg L−1) and elevated CO2 (31 mg L−1; ≈21,000 µatm) for 58 days. Following this acclimation period, fish were subjected to three separate, yet complementary, experiments: (1) acute hypercarbia challenge of 120 mg L−1 CO2 for 1 h to quantify physiological and molecular responses; (2) hypercarbia avoidance challenge to compare CO2 agitation and avoidance responses; and (3) swim performance challenge to quantify burst swimming performance. Acclimation to 31 mg L−1 CO2 resulted in a significant constitutive upregulation of c-fos expression in erythrocytes, combined with significant constitutive expression of hsp70 in both gill and erythrocytes, relative to controls. Largemouth bass acclimated to elevated CO2 also had a reduced glucose response (relative to controls) following an acute CO2 exposure, indicating a reduced stress response to CO2 stressors. In addition, largemouth bass acclimated to elevated CO2 conditions required 50 % higher CO2 concentrations to illicit agitation behaviors and displayed prolonged burst swimming abilities in high CO2 environments relative to controls. Together, results demonstrate that largemouth bass exposed to chronic hypercarbia may possess a physiological advantage during periods of elevated CO2 relative to naïve fish, which may permit increased performance in hypercarbia.

When confined in pairs, juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinate fish exhibit characteristic physiological changes including reduced growth rates and chronically elevated plasma cortisol concentrations. We hypothesized that alterations in protein metabolism contribute to the reduced growth rate of socially stressed trout, and predicted that subordinate trout would exhibit reduced rates of protein synthesis coupled with increases in protein degradation. Protein metabolism was assessed in dominant and subordinate fish after 4 days of social interaction, and in fish that were separated after 4 days of interaction for a 4 days recovery period, to determine whether effects on protein metabolism recovered when social stress was alleviated. Protein metabolism was assessed in liver and white muscle by measuring the fractional rate of protein synthesis and markers of protein degradation. In the white muscle of subordinate fish, protein synthesis was inhibited and activities of the ubiquitin-proteasome pathway (UPP) and the autophagy lysosomal system (ALS) were elevated. By contrast, the liver of subordinate fish exhibited increased rates of protein synthesis and activation of the ALS. When allowed to recover from chronic social stress for 4 days, differences in protein metabolism observed in white muscle of subordinate fish during the interaction period disappeared. In liver, protein synthesis returned to baseline levels during recovery from social stress, but markers of protein degradation did not. Collectively, these data support the hypothesis that inhibition of muscle protein synthesis coupled with increases in muscle protein breakdown contribute to the reduced growth rates of subordinate rainbow trout.

Mammals and birds adapt to prolonged fasting by mobilizing fat stores and minimizing protein loss. This strategy ends with an increase in protein utilization associated with behavioural changes promoting food foraging. Using the Zucker rat as a model, we have investigated the effect of severe obesity on this pattern of protein loss during long-term fasting. Two interactions between the initial adiposity and protein utilization were found. First, protein conservation was more effective in obese than in lean rats: fatty rats had a three times lower daily nitrogen excretion and proportion of energy expenditure deriving from proteins, and a lower daily protein loss in various muscles. This phase of protein sparing is moreover nine times longer in the fatty rats. Second, obese animals did not show the late increase in nitrogen excretion that occurred in their lean littermates. Total body protein loss during starvation was larger in fatty rats (57% versus 29%) and, accordingly, total protein loss was greater in their muscles. At the end of the experiment, lean and obese rats had lost 98% and 82%, respectively, of their initial lipid reserves, and fatty rats still had an obese body composition. These results support the hypothesis that in severely obese humans and animals a lethal cumulative protein loss is reached long before the exhaustion of fat stores, while the phase of protein conservation is still continuing. In contrast, in lean rats, survival of fasting seems to depend on the availability of lipid fuels. The data also suggest that accumulation of too much fat in wild animals is detrimental for survival, because it eliminates the late phase of increase in nitrogen excretion that is linked to a food foraging behaviour anticipating a lethal depletion of body reserves.

To investigate the proximate influence of a changing food availability on the seasonal fattening of migratory birds, garden warblers (Sylvia borin) following postnuptial moult were food restricted once a week. Body mass, food intake, plasma hormone and metabolite levels were measured and compared to birds which always had ad libitum food access. The food-restricted birds increased their body mass significantly earlier than the controls. The accelerated fattening was initially not accompanied by hyperphagia and may be due to either an increased food utilisation efficiency or a reduced metabolic rate. An increase of basal glucagon and corticosterone and a decrease of insulin levels prior to fattening were not significant, however, they resulted in a significant decrease of the insulin:glucagon ratio. This ratio was also lower in food-restricted birds than in control birds and may account for the difference in the fattening progress. We conclude that seasonal fattening may be stimulated by a catabolic impulse which could be imposed in free-living birds by a decrease of food availability and/or by an increase of energy expenditure. A negative energy balance is hypothesised to be a common proximate factor affecting migratory as well as winter fattening.

The plasma levels of four osmoregulatory hormones and their target ion-transport systems in the lower intestines of the domestic fowl were determined in order to elucidate their interrelationship and their setpoints in relation to NaCl intake. White Plymouth Rock hens were adapted to six intake levels of NaCl (0.20±0.02–24.7±1.9 mmoles Na+·kg bw−1·day−1) for 6 weeks. The Na+ absorption and the Cl− secretion of colon and coprodeum were characterized in vitro by the effects of hexoses, amino acids, amiloride, and theophylline on the short-circuit current (SCC) and electrical potential difference (PD). The NaCl-conserving system of the adult chicken is set at low intake levels of NaCl as the 80% range (quantitized by non-linear, logistic regression analyses) of the change in the plasma [ALDO], the amiloride-inhibitable Na+ absorption of coprodeum and colon (Δ SCC), occurred from 0.18 to 2.3, from 0.9 to 4.3, and from 1.2 to 7.3 mmoles Na+·kg bw−1·day−1, respectively. These results demonstrate that the amiloride-inhibitable Na+ absorption of coprodcum is more closely linked to plasma [ALDO] than that of colon. The aminoacid-Na+ coabsorption of colon increased over exactly the same range of Na+ intake as the colonic amiloride-inhibitable Na+ absorption decreased, whereas the hexose-Na+ coabsorption increased at higher levels of Na+ intake, from 2 to 11 mmoles Na+·kg bw−1·day−1. Both these Na+ absorption types had reached their maximums at 24.7 mmoles Na+·kg bw−1·day−1, whereas the plasma [AVT] and plasma [PRL], although significantly increased, apparently had not; their 80% range of change occurred from 9.9 to 99 mmoles Na+·kg bw−1·day−1, and the main changes in plasma osmolity were predicted to occur from 5.4 to 107 mmoles Na+·kg bw−1·day−1. These results suggest that these colonic and hormonal variables conserve osmotically-free water and operate at high NaCl intake. The theophylline-induced colonic Cl− secretion did not change with NaCl intake, whereas the stimulation of SCC in coprodeum decreased with increasing NaCl intake: The main change occurred between 0 and 3.2 mmoles Na+·kg bw−1·day−1. Thus, all ion-transport capacity disappears in coprodeum with increased dietary NaCl intake, whereas colon maintains its ion-transport capacity (although the nature of the Na+ transport changes). It is suggested that hormones defending the extracellular volume and composition are regulated close to zero input and output of both NaCl and water, regardless of whether they are NaCl conserving or free-water conserving. Therefore, changes in their stable plasma concentrations occur at the extremes of tolerable range of NaCl intake.