Journal of Experimental Biology

Thermal tolerance and the respiratory properties of isolated red muscle mitochondria were investigated in Oreochromis alcalicus grahami from the alkaline hot-springs, Lake Magadi, Kenya. Populations of O. a. grahami were resident in pools at 42.8°C and migrated into water reaching temperatures of 44.8°C for short periods. The maximum respiration rates of mitochondria with pyruvate as substrate were 217 and 284 natom O mg−1 mitochondrial protein min−1 at 37°C and 42°C, respectively (Q10=1.71). Fatty acyl carnitines (chain lengths C8, C12 and C16), malate and glutamate were oxidised at 70–80% of the rate for pyruvate. In order to assess evolutionary temperature adaptation of maximum mitochondrial oxidative capacities, the rates of pyruvate and palmitoyl carnitine utilisation in red muscle mitochondria were measured from species living at other temperatures: Notothenia coriiceps from Antarctica (-1.5 to +1°C); summer-caught Myoxocephalus scorpius from the North Sea (10–15°C); and Oreochromis andersoni from African lakes and rivers (22–30°C). State 3 respiration rates had Q10 values in the range 1.8–2.7. At the lower lethal temperature of O. andersoni (12.5°C), isolated mitochondria utilised pyruvate at a similar rate to mitochondria from N. coriiceps at 2.5°C (30 natom O mg−1 mitochondrial protein min−1). Rates of pyruvate oxidation by mitochondria from M. scorpius and N. coriiceps were similar and were higher at a given temperature than for O. andersoni. At their normal body temperature (-1.2°C), mitochondria from the Antarctic fish oxidised pyruvate at 5.5% and palmitoyl-DL-carnitine at 8.8% of the rates of mitochondria from the hot-spring species at 42°C. The results indicate only modest evolutionary adjustments in the maximal rates of mitochondrial respiration in fish living at different temperatures.

We examined the seasonal variation in environmentally induced protein damage in natural populations of the intertidal mussel Mytilus trossulus. In order to compare the state of protein pools during seasonal variations in environmental temperature, we used solid-phase immunochemical analysis to quantify ubiquitin conjugate concentrations and relative levels of the stress protein hsp70. The two biochemical indices were selected for their cellular roles in irreversible and reversible protein denaturation, respectively. Proteins that are ubiquitinated are irreversibly damaged and are degraded by intracellular proteases; stress proteins act as molecular chaperones to re-fold thermally denatured proteins and, thus, indicate degrees of reversible protein damage. Comparisons involved mussels collected in February and August from two study sites: an intertidal site which subjected animals to a wide range of body temperatures (from approximately 10 to 35 °C in summer), and a subtidal site where animals remained submerged throughout the tidal cycle. Our results show that quantities of ubiquitin conjugates and hsp70 were greater in gill tissue from summer-collected mussels than in gills of winter-collected specimens. Ubiquitin conjugate and hsp70 levels were also greater in mussels collected from an intertidal location than in mussels from a submerged population. Our results show that the high summer temperatures normally experienced in the field are sufficient to cause increased denaturation of cellular proteins. Despite increases in the concentrations of heat shock proteins in summer-acclimatized mussels, elevated levels of irreversibly denatured, i.e. ubiquitinated, proteins were still observed, which indicates that the heat shock response may not be able to rescue all heat-damaged proteins. The energy costs associated with replacing heat-damaged proteins and with maintaining the concentrations and activities of heat shock proteins may contribute substantially to cellular energy demands. These increased energy demands may have an impact on the ecological energetic relationships of species, e.g. in the allocations of energy for growth and reproduction, and, as a consequence, may contribute to determining their distribution limits.

The abundance, distribution and oxidative capacities of mitochondria have been investigated in the red pectoral fin adductor muscles of fish (Order Perciformes) that use a predominantly labriform style of swimming. Mediterranean Sea species from the families Labridae, Serranidae, Sparidae and Antarctic Nototheniidae and non-Antarctic Nototheniidae and Channichthyidae were studied. Sub-Antarctic species from the Beagle Channel, Tierra del Fuego, included the pelagic haemoglobin-less icefish (Champsocephalus esox) and the róbalo (Eleginops maclovinus), which occurs as far north as 35°S. In Champsocephalus esox, the mitochondrial volume density of red muscle was 0.51 and mitochondrial cristae surface density (43.9 μm2μm−3) was higher than reported for Antarctic icefishes. In the red-blooded, active pelagic or semi-pelagic species, mitochondrial volume density was within the range 0.27–0.33 regardless of habitat temperature. Amongst less active demersal species, mitochondrial volume density ranged from 0.29–0.33 in polar species to 0.08–0.13 in Mediterranean species. In Antarctic species and Champsocephalus esox, myofibrils occurred in ribbons or clusters one fibril thick entirely surrounded by mitochondria. The volume density of intracellular lipid droplets was not correlated with activity patterns or habitat temperature. In a comparison of Eleginops maclovinus caught in summer (approximately 10°C) and winter (approximately 4°C), mitochondrial volume density did not differ, whereas the surface density of mitochondrial clusters was higher in summer fish. The temperature-dependence of the state 3 respiration rate of isolated mitochondria with pyruvate as substrate was described by a single quadratic relationship for all species, indicating no significant up-regulation of the maximum rate of oxygen uptake per milligram mitochondrial protein in Antarctic species. Our results support the conclusion that increasing the volume and surface density of mitochondrial clusters is the primary mechanism for enhancing the aerobic capacity of muscle in cold-water fish.

Protein synthesis is a fundamental and energetically expensive physiological process in all living organisms. Very few studies have examined the specific challenges of manufacturing proteins at low ambient temperatures. At high southern latitudes, water temperatures are continually below or near freezing and are highly stable, while food availability is very seasonal. To examine the effects of low temperature and a highly seasonal food supply on protein metabolism, we have measured wholebody protein synthesis, RNA concentrations, RNA:protein ratios and RNA translational efficiencies in the Antarctic limpet Nacella concinna at four times of the year. From summer to winter, protein synthesis rates decreased by 52%, RNA concentrations decreased by 55% and RNA:protein ratios decreased by 68%, while RNA translational efficiencies were low and very variable. Protein synthesis rates in N. concinna approached those measured in temperate mussels, while RNA:protein ratios were considerably higher than in temperate species. Interspecific comparisons show that species living at low temperatures have elevated RNA:protein ratios, which are probably needed to counteract a thermally induced reduction in RNA translational efficiency. Calculations using theoretical energetic costs of protein synthesis suggest that Antarctic species may allocate a larger proportion of their metabolic budget to protein synthesis than do temperate or tropical species.

In teleosts, the proliferation of myogenic progenitor cells is required for muscle growth and nuclear turnover. We measured the cell cycle and S-phase duration of myogenic cells in the fast myotomal muscle of two closely related Harpagifer species by cumulative S-phase labelling with 5-bromo-2′-deoxyuridine (BrdU). Harpagifer antarcticus is a stenothermal species from the Antarctic peninsula (experiencing temperatures of -2°C to +1°C) and Harpagifer bispinis is a eurythermal species from the Beagle Channel, Tierra del Fuego (living at +4°C in winter and up to 11°C in summer). Specific growth rates in the adult stages studied were not significantly different from zero. Myogenic progenitor cells were identified using an antibody against c-met. Seventy-five percent of the c-met+ve cells were in a proliferative state in both species. Cell cycle time was 150 h at 5°C and 81.3 h at 10°C in H. bispinis (Q10=3.4). Cell cycle duration was 35% shorter in H. antarcticus at 0°C (111 h) than in H. bispinis at 5°C. The predicted cell cycle time for H. bispinis at 0°C(based on the Q10 relationship) was 277 h, which was more than double that measured for the Antarctic species at this temperature. The results obtained are compatible with an evolutionary adjustment of cell cycle time for function at low temperature in the Antarctic species.

Insects have long been known to excrete toxins via the Malpighian (renal) tubules. In addition, exposure to natural or synthetic toxins is commonly associated with increases in the activity of detoxification enzymes such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs). We examined the links between mechanisms for detoxification and excretion in adult Drosophila melanogaster using functional assays and measurements of changes in gene expression by quantitative reverse transcriptase PCR in response to dietary exposure to compounds known to alter activity or gene expression of P450s and GSTs. Dietary exposure to phenol, which alters gene expression for multiple GSTs after seven to 10 generations, was also associated with an increase (more than twofold) in secretion of the organic anion methotrexate (MTX) by isolated tubules. Dietary exposure to the insecticide synergist piperonyl butoxide (PBO) was associated with reduced expression of two P450 genes (Cyp4e2, Cyp4p1) and two GST genes (GstD1, GstD5) in the tubules, as well as increased expression of Cyp12d1 and GstE1. Thin layer chromatographic analysis of fluid secreted by isolated tubules indicated that dietary exposure to PBO resulted in increased levels of an MTX metabolite. In addition, exposure to PBO altered the expression of transporter genes in the tubules, including a Drosophila multidrug resistance-associated protein, and was associated with a 73% increase in MTX secretion by isolated tubules. The results suggest that exposure of Drosophila to toxins evokes a coordinated response by the Malpighian tubules, involving both alterations in detoxification pathways as well as enhanced transport.

Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome.

We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin,suggesting the presence of receptor-mediated endocytosis. After endocytosis,hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium–mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro.

The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.

O2 uptake was determined for periods of 23–46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10°C. These media were replaced by artificial lakewater media (ALW) containing 0·20–6 mM Na and O2 uptake was again determined for periods of 24–66 h. During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0·1–15·5% (mean 6·4 (15)14·4% s.D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22·0–66·7%, but some of this increase may have been due to locomotor activity. The calculated values for thermodynamic work involved in ion transport were 0·056–0·268 J/10 g.h at 10°C, or 1·5–7·2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. ± 7 %). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.

The sodium content of the freshwater prawn Palaemonetes antennarius and the rates of influx and efflux of sodium in fresh water and a variety of other media are described. The greater part of the influxes in all media is due to active uptake. The relationship between active uptake and the external and internal concentrations is described and it is concluded that the animal is living close to its viable limits of dilution. The greater part of the efllux in fresh water is due to loss in the urine.

The dynamics of osmoregulation in an ideal semi-permeable animal are discussed. The theoretical minimum osmotic work is evaluated in terms of the surface area of the animal, its permeability and the concentrations of the blood, urine and external medium. It is shown that : (a) The most important means whereby a marine animal entering brackish water can reduce the strain upon its osmoregulatory mechanisms is by reducing the concentration of its blood. (b) In a brackish-water animal the production of urine hypotonic to. the blood has only a very small effect upon the osmotic work. (c) In a fresh-water animal the reduction of the urine concentration to the point at which it is isotonic with the medium can reduce the osmotic work by as much as 90 % ; but even a moderate reduction of the urine concentration, so that the urine is hypotonic to the blood but many times more concentrated than the medium, greatly reduces the osmotic work and is compatible with high osmoregulatory efficiency. These conclusions are discussed with reference to some fresh-water animals.