The protein synthesis machinery operates at the same expense in eurythermal and cold stenothermal pectinids

Translationally active cell-free systems from gills of the Antarctic scallop Adamussium colbecki and the European scallop Aequipecten opercularis were developed, characterised, and optimised for an analysis of translational capacity. The aim was to determine the energetic cost of protein synthesis in the in vitro cell-free system by directly measuring the required energy equivalents in the lysates. Protein synthesis rate in assays conducted with lysates of A. colbecki (1.029+/-0.061 micromol Phe min(-1) at 15 degrees C; Phe=phenylalanine) were higher compared with lysates of A. opercularis (0.087+/-0.013 micromol Phe min(-1) at 15 degrees C and 0.156+/-0.023 micromol Phe min(-1) at 25 degrees C). This can in part be attributed to the naturally occurring higher RNA content in lysates of A. colbecki (0.883+/-0.037 mg RNA mL(-1) lysate) compared with A. opercularis (0.468+/-0.013 mg RNA mL(-1) lysate). There was no significant difference in the energetic costs of protein synthesis in cell-free systems of gill lysates of the cold stenothermal A. colbecki with 4.3+/-0.7 energy equivalents per peptide bond formed and the eurythermal A. opercularis with 5.6+/-0.6 energy equivalents, indicating that there are no differences in the efficiency of the translation machinery. The energetic costs specified for protein synthesis correspond with the generally accepted theoretical value of four energy equivalents per peptide bond formed, especially in gill lysates of A. colbecki, whereas the value for gill lysates of A. opercularis was slightly higher.     

Molecular Data from the 16S rRNA Gene for the Phylogeny of Pectinidae (Mollusca: Bivalvia)

The phylogenetic relationships among the species belonging to the family Pectinidae are still an issue of debate. The mitochondrial DNA sequences from the large ribosomal RNA gene may be of great value for systematic and phylogenetic studies within families. Partial sequences of the 16S rRNA gene were obtained for the scallop species Adamussium colbecki, Aequipecten opercularis, Chlamys glabra, C. islandica, C. varia, and Pecten jacobeus and compared with the published sequence of Pecten maximus. The present molecular data show that Chlamys are polyphyletic and do not support the assignment of these species to the two subfamilies Chlamydinae and Pectininae. Moreover, the minimal genetic distance between P. maximus and P. jacobeus suggests that they could belong to the same species. Received: 24 May 1999 / Accepted: 1 September 1999     

Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae).

The internal transcribed spacer (ITS) region of the ribosomal DNA from the European scallops Aequipecten opercularis, Mimachlamys varia, Hinnites distortus, and Pecten maximus was PCR amplified and sequenced. For each species, three or five clones were examined. The size ranged between 636 and 713 bp (ITS1, 209-276 bp; 5.8S rRNA gene, 157 bp; ITS2, 270-294 bp) and GC content ranged between 47 and 50% (ITS1, 43-49%; 5.8S rRNA gene, 56-57%; ITS2, 44-49%). Variation within repeats was minimal; only clones from M. varia and P. maximus displayed a few variable sites in ITS2. Among scallops, including Chlamys farreri whose ITS sequence appears in databases, significant variation was observed in both ITS1 and ITS2. Phylogenetic analysis using ITS1, ITS2, or both spacer sequences always yielded trees with similar topology. Aequipecten opercularis and P. maximus grouped in one clade and the other three scallops (C. farreri, M. varia, and H. distortus) in another, where M. varia and H. distortus are the more closely related species. These results provide new insights into the evolutionary relationships of scallop species and corroborate the close evolutionary relationship between the tribes Aequipectinini and Pectinini previously deduced from 18S rDNA sequences.     

Mode of locomotion places selective pressures on Antarctic and temperate labriform swimming fish

The physiological responses to exercise and stress of the Antarctic labriform swimmer Pagothenia borchgrevinki were compared to the temperate labriform swimmers Notolabrus celidotus and Notolabrus fucicola. Basic swimming characteristics were very similar amongst the three species with P. borchgrevinki showing a reduced capacity for exercise. P. borchgrevinki showed large increases in haematocrit (Hct) following exercise that were not seen in the temperate species. Lactate dehydrogenase (LDH) activities were high in the white myotomal muscle from the Antarctic fish, with a distinct indication of metabolic cold adaptation in this enzyme. Nevertheless, although the temperate fish showed elevated muscle lactate concentrations following either exercise or electrical stimulation the Antarctic fish did not. The data suggest that poor anaerobic performance of white muscle is associated with the labriform mode of locomotion.     

Defenses against oxidative stress in the Antarctic scallop Adamussium colbecki and effects of acute exposure to metals

Since a general pathway of toxicity induced bypollutants is the enhancement of reactive oxygenspecies, biochemical responses associated withvariations in the antioxidant cellular system havebeen often proposed as biomarkers ofpollutant-mediated toxicity associated with oxidativestress. Antarctic organisms live in an extremeenvironment characterized by low water temperature,high level of dissolved oxygen, presence of ice andstrong seasonal changes in light intensity andavailability of food, conditions which could influenceboth the formation of reactive oxygen species and themechanisms for their removal. In this respect andconsidering the utility of this as a key species formonitoring marine Antarctic environment it was ofinterest to investigate the antioxidant defense systemof the scallop Adamussium colbecki.The parameters examined in the digestive gland of thescallop were the concentration of glutathione and theactivity of several glutathione dependent andantioxidant enzymes (glyoxalase I and II, glutathioneS-transferases, glutathione peroxidases, glutathionereductase, catalase, superoxide dismutase). Very highlevels of catalase suggest a possible adaptation toAntarctic extreme conditions, while the highactivities of glutathione S-transferases are moreprobably related to the feeding behavior of Pectinids.Enzymes from Adamussium colbecki generallyappeared to be active at low temperatures but, with afew exceptions, their activities increased with risingtemperature. Exposure of A. colbecki tosublethal concentrations of Cu or Hg resulted in asignificant reduction in the levels of totalglutathione and in the activity of catalase andglutathione S-transferases. Antioxidant responses ofA. colbecki could represent a useful tool inassessing the biological impact of environmentalpollutants in the Antarctic ecosystems.     

An in situ study of predator aggregations on scallop (Pecten maximus (L.)) dredge discards using a static time-lapse camera system

The impact of demersal fishing gears on benthic habitats and species has been the subject of much attention recently, and suggestions have been made that scavenging epifaunal species may benefit at the population level from the additional food source provided by discards. This paper investigates some aspects of this process, including the relative attractiveness to predators of different discard species, and the role of damage in scavenger attraction. A time-lapse video system with a 1000 m long cable was positioned in an area closed to fishing, adjacent to the most heavily fished scallop (Pecten maximus) ground in the Irish Sea. A variety of undamaged and damaged by-catch animals were positioned in front of the camera, and the subsequent predator aggregations investigated. Densities of scavenger species up to 200 times that of the background population were observed, and aggregations of some species persisted for up to 3 days. The most frequently recorded scavengers, and therefore presumably those species most likely to benefit from discards as a food source, were: Asterias rubens L., Astropecten irregularis (Pennant), Liocarcinus spp Stimpson, Pagurus spp Fabricius and Callionymus lyra L. Predator attraction to apparently undamaged queen scallops, Aequipecten opercularis (L.), was almost as high as to damaged A. opercularis. Of all the prey species studied, queen scallops were the most attractive to scavengers. A directional relationship was found between the ambient water current and the arrival of the starfish, Asterias rubens.     

A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species

Antarctic fish have a high polyunsaturated lipid content and their muscle cells have a high mitochondria density suggesting that Antarctic fish are under greater oxidative stress than temperate water fish. To test this hypothesis, the plasma concentrations of the antioxidant vitamins E and C were measured in two Antarctic fish species, Pagothenia borchgrevinki and Trematomus bernacchii, and compared with the plasma concentrations of these vitamins in two New Zealand temperate water fish species, blue cod (Parapercis colias) and banded wrasse (Notolabrus fucicola). Neither vitamin is known to be synthesised in fish and so must be obtained from the diet. The plasma from both Antarctic fish species had vitamin E concentrations five to six times higher than those found in the two temperate water fish species. However, significantly higher levels of vitamin C were only found in the plasma of T. bernacchii, a benthic Antarctic fish. The average level of vitamin C in the plasma of the cryopelagic P. borchgrevinki was approximately one-third that of T. bernacchii. The T. bernacchii plasma yielded a high range of vitamin C values, possibly reflecting differences in nutritional status among the animals captured. No beta-carotene was found in any of the fish plasma samples studied. The data suggest that even though Antarctic fish live at -1.5 degrees C they may be exposed to greater metabolic stress from free radical mediated oxidation than temperate water species.     

Recovery from exhausting exercise in an Antarctic fish,Pagothenia borchgrevinki

Summary Pagothenia borchgrevinki, an Antarctic teleost fish was swum to exhaustion and changes in lactic acid levels and blood haematocrit were monitored during recovery. The fish did not perform well at high swimming speeds due to an inability of the white myotomal muscle to produce ATP by anaerobic glycolysis. Consequently, low levels of lactic acid were produced which were fairly rapidly broken down. Haematocrit values were low in non-exercised fish, and these increased by over 100% during exercise, falling back to control levels over many hours. This is probably related to the increased oxygen demand during exercise.     

Identification of four scallop species using PCR and restriction analysis of the ribosomal DNA internal transcribed spacer region

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA region spanning the 5.8S RNA gene and the 2 flanking internal transcribed spacers (ITSs) was performed to establish DNA-based molecular markers for the identification of the scallops Aequipecten opercularis, Chlamys distorta, Mimachlamys varia, and Pecten maximus. Chlamys distorta was distinguished simply by ITS size. Species-specific restriction patterns were found with the restriction enzyme AluI, and also with SmaI for A. opercularis and M. varia. When ITS sizes and the RFLPs obtained with SmaI were combined, the 4 scallops were also differentiated. Additional species-specific RFLPs were revealed after ITS-2 PCR amplification and subsequent digestion with Hsp92II. Using this marker, canned scallops were identified. Thus this work provides a simple, reliable, and rapid method for the identification of scallops that can be used when species-specific morphologic characteristics are removed or when specimens are small in size.     

Unusual trophic strategies of Hydractinia angusta (Cnidaria, Hydrozoa) from Terra Nova Bay, Antarctica

Hydractinia angusta Hartlaub, 1904 has been recorded at Terra Nova Bay (Ross Sea, Antarctica) as epizoic on shells of the Antarctic scallop Adamussium colbecki. The species can exploit different trophic resources: first, polyps are able to detach and ingest tube feet and pedicellariae from the sea urchins Sterechinus neumayeri, grazing on the scallop shell, and second, they also eat masses of benthic diatoms settled among the hydrorhiza of the colony. The particular relationship observed between the hydroid and one of the most common Antarctic sea urchins may prevent or reduce the damage to A. colbecki shells, otherwise caused by the grazing of sea urchins on the algal film of the upper valve of the scallops. H. angusta is the first known species of hydroid that exploits prey several times its own size and the second that does not ingest entire prey but portions of them. The use of benthic diatoms as a food resource has previously been documented for the sub-Antarctic marine hydroid Silicularia rosea. Accepted: 20 December 1999     

PDC activity and acetyl group accumulation in skeletal muscle during prolonged exercise.

Seven subjects cycled to exhaustion [58 +/- 7 (SE) min] at approximately 75% of their maximal oxygen uptake (VO2max). Needle biopsy samples were taken from the quadriceps femoris muscle at rest, after 3, 10, and 40 min of exercise, at exhaustion, and after 10 min of recovery. After 3 min of exercise, a nearly complete transformation of the pyruvate dehydrogenase complex (PDC) into active form had occurred and was maintained throughout the exercise period. The total in vitro activated PDC was unchanged during exercise. The muscle concentration of acetyl-CoA increased from a resting value of 8.4 +/- 1.0 to 31.6 +/- 3.3 mumol/kg dry wt at exhaustion and that of acetylcarnitine from 2.9 +/- 0.7 to 15.6 +/- 1.6 mmol/kg dry wt. This was accompanied by corresponding decreases in reduced CoA (CoASH) from 45.3 +/- 3.1 to 25.9 +/- 3.1 mumol/kg dry wt and in free carnitine from 18.8 +/- 0.7 to 5.7 +/- 0.5 mmol/kg dry wt. Acetyl group accumulation, in the form of acetyl-CoA and acetylcarnitine, was maintained throughout exercise to exhaustion while the glycogen content decreased by 90%. This suggests that availability of acetyl groups was not limiting to exercise performance despite the nearly total depletion of the glycogen store. The increased acetyl-CoA-to-CoASH ratio during exercise caused inhibition of neither the PDC transformation nor the calculated catalytic activity of active PDC.     

A satellite DNA containing CENP-B box-like motifs is present in the antarctic scallop Adamussium colbecki

The DNA of the Antarctic scallop Adamussium colbecki was found to contain a highly repeated sequence identifiable upon restriction with endonuclease BglII. The monomeric unit - denominated pACS (about 170bp long) - was cloned. Southern blot hybridization yielded a ladder-like banding pattern, indicating that the repeated elements are tandemly arranged in the genome and therefore represent a sequence of satellite DNA.Sequence analysis of five different clones revealed the presence of various subfamilies, some of which showed a high degree of divergence. In each clone, regions homologous to the mammalian CENP-B box were observed. A region homologous to the CDEIII centromeric sequence of yeast was also found in one of the clones. These observations suggest a relationship of the pACS family to the centromeric area in A. colbecki.     

Muscle glycogen, lactate and glycerol-3-phosphate concentrations of larval and young adult lampreys in response to exercise

When stimulated, the ammocoetes (larvae) of Geotria australis swim continuously at a moderate rate for only approximately 20 min, whereas the downstream migrants (young adults) of this species did not become exhausted following similar swimming activity over the same period. Mean concentrations of muscle glycogen in ammocoetes declined during exercise, but returned to resting levels within 30 min of recovery, whereas those in young adults changed little during the corresponding periods. Moreover, muscle lactate concentrations of ammocoetes rose markedly during exercise and the first 30 min of recovery, before declining significantly, while those of young adults remained similar during and immediately after exercise. Calculations, using the glycogen and lactate concentrations immediately after exercise, suggest that during exercise glycogen is, to some extent, utilised anaerobically (approx. 24%) by ammocoetes, but only aerobically by young adults. Furthermore, since young adults used only a small amount of glycogen, they presumably metabolised triacylglycerol aerobically to produce energy. Muscle glycerol-3-phosphate levels were far higher prior to and immediately after exercise in downstream migrants than in ammocoetes and then declined precipitously. The above trends in muscle glycogen and lactate of larval G. australis parallels, to some degree, those recorded by other workers for upstream migrant Petromyzon marinus that had been exercised to exhaustion.     

Lactate and glycogen metabolism during and after exercise in the lizardSceloporus occidentalis

Summary Lactate removal and glycogen replenishment were studied in the lizardSceloporus occidentalis following exhaustion at 35°C. Whole body lactate concentrations and oxygen consumption were measured inSceloporus at rest, after 2 min vigorous exercise and at intervals during a 150 min recovery period. Lactate concentrations peaked at 2.2 mg/g (24 mM) after exercise and returned to resting levels after 90 min. Oxygen consumption returned to resting rates after 66 min. In a second set of experiments, glycogen and lactate concentrations of liver, hindlimb and trunk musculature were measured over the same time periods of exercise and recovery. The decrease in muscle glycogen following exercise was identical (mg/g) to the increase in muscle lactate, and the stoichiometric and temporal relationships between lactate removal and glycogen replenishment during the recovery period were also similar. Glycogen replenishment was rapid (within 150 min) and complete in fastedSceloporus. Dietary supplement of carbohydrate during 48 h of recovery led to supercompensation of glycogen stores in the muscle (+66%) and liver (+800%). The changes were similar to the seasonal differences measured inSceloporus from the field.     

Hormonal responses to exercise during moderate cold exposure in young vs. middle-age subjects.

The influence of moderate cold exposure on the hormonal responses of atrial natriuretic factor (ANF), arginine vasopressin (AVP), catecholamines, and plasma renin activity (PRA) after exhaustive exercise was studied in 9 young and 10 middle-aged subjects. Exercise tests were randomly performed in temperate (30 degrees C) and cold (10 degrees C) environments. Heart rate, oxygen consumption, and peripheral arterial blood pressure were measured at regular intervals. Blood samples were collected before and immediately after exercise at 30 or 10 degrees C. Plasma sodium and potassium concentrations as well as hemoglobin and hematocrit were measured, and the change in plasma volume was calculated. At rest and during exercise, oxygen consumption was similar during exposure to both temperate and cold temperatures. During submaximal exercise intensities, the rise in heart rate was blunted while the increase in systolic blood pressure was significantly greater at 10 than at 30 degrees C. The increases in plasma sodium and potassium concentrations after exhaustion were similar between environments, as was the decrease in plasma volume. In both groups, all plasma hormones were significantly elevated postexercise, with the AVP response similar at 10 and 30 degrees C. However, the norepinephrine and ANF responses were significantly greater while the PRA response was significantly reduced at 10 degrees C. In the middle-aged subjects the epinephrine response to exercise was higher at 10 than at 30 degrees C. The greater ANF and reduced PRA responses to exercise in the cold may have resulted from central hemodynamic changes caused by cold-induced cutaneous vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate dehydrogenases in Antarctic and temperate fish species

1. The kinetics of lactate dehydrogenase (both forward and back reaction) in cardiac and skeletal muscle of an Antarctic teleost have been compared with a temperate teleost of comparable morphology and ecology. 2. In both species the forward reaction (pyruvate to lactate) is maximally activated at 2.5-4 mM pyruvate and inhibited above this level. 3. The Michaelis constant (Km) for pyruvate is not significantly different between muscle types or between species when measured at their normal environmental temperature. 4. Km for pyruvate varies with temperature in a positive direction. 5. The back reaction (lactate to pyruvate) is maximally activated by 12-16 mM lactate but only in skeletal muscle of the antarctic species is there inhibition above this level. 6. The Km for lactate is significantly (P less than 0.05) lower in the Antarctic fish cardiac muscle. 7. While the two species are morphologically and ecologically similar, differences at the biochemical level are discussed with respect to environmental temperature range and conservation of enzymic characteristics.     

Caffeine mouth rinse enhances performance,fatigue tolerance and reduces muscle activity during moderate-intensity cycling

We investigated the effects of caffeine mouth rinse on endurance performance, muscle recruitment (i.e., electromyographic activity of the vastus lateralis and rectus femoris), rating of perceived effort and heart rate. Twelve physically-active healthy men cycled at 80% of their respiratory compensation point until task failure. The participants rinsed their mouths for 10 seconds with placebo (PLA, 25 mL of a solution composed of non-caloric mint essence) or caffeine (CAF, 25 mL of 1.2% of anhydrous caffeine concentration with non-caloric mint essence) every 15 minutes of exercise. Time until exhaustion increased 17% (effect size = 0.70) in CAF compared to PLA (p = 0.04). The wavebands of low-frequency electromyographic activity (EMG) of the vastus lateralis and rectus femoris was lower in CAF group than PLA at 50% of the time until exhaustion (p = 0.04). The global EMG signal was lower in CAF group than PLA at 100% of the time until exhaustion (p = 0.001). The rating of perceived effort pooled was higher in CAF mouth rinse (p = 0.001) than PLA group. No effect was found on the heart rate between the groups (p > 0.05). Caffeine mouth rinse increases endurance performance, rating of perceived effort and decreases muscle activity during a moderate-intensity exercise.     

Depletion of high energy phosphates implicates post-exercise mortality in carp and trout; an in vivo 31P-NMR study

As in vivo 31P-Nuclear Magnetic Resonance spectroscopy is currently the state of the art method to measure continuously intracellular pH (pH(i)) and energy status of muscle tissue, we used this method to study the recovery from exhaustive exercise. The biochemical changes during recovery are not well understood and it was suggested that post-exercise mortality could be caused by low pH(i); other studies however indicate that energy depletion might be more important. To analyse the mechanism of post-exercise recovery pH(i), ATP, P(i), and PCr must be measured at the same time, which is possible using in vivo 31P-NMR. Common carp and rainbow trout of about 100 g were exercised to exhaustion in a swim tunnel. After swimming 10 h at 1.5 body lengths (BL)/s (aerobic control), 50% of the fish were forced to swim at 6 BL/s until exhaustion. Recovery of energy rich phosphates was found to be faster in carp (1.2-1.9 h) than in trout (1.5-2.3 h). The same applied for the recovery from acidosis, which took 1.75 h in carp and 5.75 h in trout. In parallel experiments the energy phosphates and lactate levels were measured in liver, red muscle, and white muscle. Exhaustion caused a significant drop in the energy status of red and white muscle tissue of trout and carp (corroborates NMR data), while no change at all was observed in liver tissue. The lactate levels were increased in the muscle but not in liver and blood. While all experimental animals looked healthy after exhaustion, 40-50% of the carp as well as trout died during the recovery phase. The energy status of those individuals measured by 31P-NMR was much lower than that of the survivors, while in contrast there was no difference in pH(i). Thus, it appears that not acidosis but depletion of high energy phosphates disabled muscle function and therefore may have been the cause of death of the non-survivors.     

Depletion of high energy phosphates implicates post-exercise mortality in carp and trout; an in vivo 31P-NMR study.

As in vivo 31P-Nuclear Magnetic Resonance spectroscopy is currently the state of the art method to measure continuously intracellular pH (pH(i)) and energy status of muscle tissue, we used this method to study the recovery from exhaustive exercise. The biochemical changes during recovery are not well understood and it was suggested that post-exercise mortality could be caused by low pH(i); other studies however indicate that energy depletion might be more important. To analyse the mechanism of post-exercise recovery pH(i), ATP, P(i), and PCr must be measured at the same time, which is possible using in vivo 31P-NMR. Common carp and rainbow trout of about 100 g were exercised to exhaustion in a swim tunnel. After swimming 10 h at 1.5 body lengths (BL)/s (aerobic control), 50% of the fish were forced to swim at 6 BL/s until exhaustion. Recovery of energy rich phosphates was found to be faster in carp (1.2-1.9 h) than in trout (1.5-2.3 h). The same applied for the recovery from acidosis, which took 1.75 h in carp and 5.75 h in trout. In parallel experiments the energy phosphates and lactate levels were measured in liver, red muscle, and white muscle. Exhaustion caused a significant drop in the energy status of red and white muscle tissue of trout and carp (corroborates NMR data), while no change at all was observed in liver tissue. The lactate levels were increased in the muscle but not in liver and blood. While all experimental animals looked healthy after exhaustion, 40-50% of the carp as well as trout died during the recovery phase. The energy status of those individuals measured by 31P-NMR was much lower than that of the survivors, while in contrast there was no difference in pH(i). Thus, it appears that not acidosis but depletion of high energy phosphates disabled muscle function and therefore may have been the cause of death of the non-survivors.