Substrate and enzyme profile of fast and slow skeletal muscle fibers in rhesus monkeys

Results from the Russian Cosmos program suggest that the rhesusmonkey is an excellent model for studying weightlessness-induced changes in muscle function. Consequently, the purpose of this investigation was to establish the resting levels of selected substrateand enzymes in individual slow- and fast-twitch muscle fibers of therhesus monkey. A second objective was to determine the effect of an18-day sit in the Spacelab experiment-support primate facility[Experimental System for the Orbiting Primate (ESOP)].Muscle biopsies of the soleus and medial gastrocnemius muscles wereobtained 1 mo before and immediately after an 18-day ESOP sit. Thebiopsies were freeze-dried, and individual fibers were isolated andassayed for the substrates glycogen and lactate and for the high-energyphosphates ATP and phosphocreatine. Fiber enzyme activity was alsodetermined for the glycolytic enzymes phosphofructokinase and lactatedehydrogenase (LDH) and for the oxidative markers 3-hydroxyacyl-CoAdehydrogenase (-OAC) and citrate synthase. Consistent with otherspecies, the fast type II fibers contained higher glycogen content thandid the slow type I fibers. The ESOP sit had no significant effects onthe metabolic profile of the slow fibers of either muscle or the fast fibers of the soleus. However, the fast gastrocnemius fibers showed asignificant decline in phosphocreatine and an increase in lactate. Also, similar to other species, the fast fibers contained significantly higher LDH activities and lower 3-hydroxyacyl-CoA dehydrogenase activities. For the muscle enzymes, the quantitatively most important effect of the ESOP sit occurred with LDH where activities increased inall fiber types postsit except the slow type I fiber of the medial gastrocnemius.

     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Effect of cadmium intoxication on glucose utilization in energy metabolism of muscles

The influence of cadmium intoxication on carbohydrate metabolism in skeletal muscles and liver of the male Wistar rats has been studied. Cadmium was administered as cadmium acetate in a dose of 0.3 mg Cd2+/kg body weight for three months. At the same time the control rats were injected with 0.9% NaCl. The animals were decapitated and samples of their skeletal muscles: the soleus muscle (composed mainly of red slow twitch fibers; ST) the gastrocnemius muscle containing two types of fibers (white fast twitch fibers FTb and red fast twitch fibers, FTa) and the liver were dissected out. In the samples of muscles, liver and serum contents of glycogen, glucose, pyruvate and lactate, as well as activities of hexokinase, pyruvate kinase and lactate dehydrogenase were measured. Intoxication of rats with cadmium for three months resulted in a reduction of glycolytic enzymes in the serum, ST and FTa muscle fibers and in the liver but did not change the activities of glycolytic enzymes in the FTb muscle fibers. The data obtained for the concentrations of glycogen in the liver and skeletal muscles suggest different mechanisms of cadmium influence on glycogen utilization in these organs.     

Enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles of hypocaloric rats.

1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 +/- 0.19, 2.68 +/- 0.65, and 1.20 +/- 0.33 nmol x min(-1) x g wet tissue wt(-1) in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 +/- 0.22, 2.00 +/- 0.57, and 0.84 +/- 0.18 nmol x min(-1) x g wet tissue wt(-1)). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fast-twitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter- and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.     

Effect of denervation on the distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition

Phosphoglycerate mutase (PGM) and creatine phosphokinase (CK) occur as three isozymes (types MM, MB and BB) in mammals and these exhibit similar transitions during skeletal muscle development. To study the influence of innervation on this transition and on the maintenance of the isozyme phenotype in mature muscle, we have determined the changes produced by sciatic neurectomy in neonatal and adult rat hindlimb muscles. In 40-day-old rats, denervation decreased both PGM and CK activity, the effect being more pronounced in the fast-twitch extensorum digitorum longus (EDL) and gastrocnemius muscles than in the slow-twitch soleus muscle. It also produced a progressive increase in the proportion of MB- and BB-PGM isozymes in EDL and gastrocnemius but not in soleus, and an increase of MB- and BB-CK isozymes in all three muscles. In 5-day-old rats, denervation prevented the developmental increase of PGM and CK activity in all three muscles. Denervation also prevented the normal decrease in the relative amounts of the MB and BB isozymes of both enzymes which occur during postnatal muscle development. These results can be explained by the different effects of denervation upon slow and fast muscles, and by the distinct distribution of PGM and CK isozymes in rat type I and II muscle fibers.     

Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.     

Isometric training of young rats — Effects upon hind limb muscles

The soleus, rectus femoris, and gastrocnemius muscles of young rats trained isometrically for 4 weeks were studied by light and electron microscopy.--The percentage of fast-twitch oxidative muscle fibers decreased at the cost of the fast-twitch glycolytic fibers in the rectus femoris muscle. The percentages of the slow-twitch oxidative fibers did not change significantly in any of the muscles studied. The changes in the areas of the muscle fibers were specific for the muscle and the fiber type and indicate geometrical rearrangements of the fibers in the trained muscles. The Z and M lines were broader in the soleus (containing about 85% slow-twitch oxidative fibers) than in the rectus femoris muscle (containing about 90% fast-twitch glycolytic fibers), while the sarcomere length and the pseudo-H zone were similar. The length of the myosin filaments appeared to be slightly shorter in the fast rectus femoris than in the slow soleus muscle.--The hypothesis on the temporal progress of muscle adaptation to training (Müller, 1974) was substantiated. Correlations between biochemical (Exner et al., 1973a) and histochemical parameters measuring the oxidative capacity were preserved during adaptation to training. The comparison of the histochemical results with the physiological data on similar animals (Exner et al., 1973a) suggests a complex relationship between the contraction time and the percentage of fast-twitch muscle fibers.     

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.     

No effects of lifelong creatine supplementation on sarcopenia in senescence-accelerated mice (SAMP8)

Oral creatine supplementation can acutely ameliorate skeletal muscle function in older humans, but its value in the prevention of sarcopenia remains unknown. We evaluated the effects of lifelong creatine supplementation on muscle mass and morphology, contractility, and metabolic properties in a mouse model of muscle senescence. Male senescence-accelerated mice (SAMP8) were fed control or creatine-supplemented (2% of food intake) diet from the age of 10 to 60 wk. Soleus and extensor digitorum longus muscles were tested for in vitro contractile properties, creatine content, and morphology at weeks 25 and 60. Both muscle types showed reduced phosphocreatine content at week 60 that could not be prevented by creatine. Accordingly, age-associated decline in muscle mass and contractility was not influenced by treatment. Aged soleus muscles had fewer and smaller fast-twitch glycolytic fibers irrespective of treatment received. It is concluded that lifelong creatine supplementation is no effective strategy to prevent sarcopenia in senescence-accelerated mice.     

Some properties of different skeletal muscle fiber types: comparison of reference bases

Several biochemical components of the white portion of the gastrocnemius (WGM), plantaris (PM), and soleus (SM) muscles of the rat and middle gluteal (MGM) muscle of the horse were compared based on wet and dry weight, protein, and total creatine concentrations ([TCr]). The water content was similar for the rat hindlimb muscles, however, the concentrations of protein, ATP, phosphocreatine (PCr), creatine, and glycogen ranked as SM less than PM less than WGM for all reference bases except total creatine. In contrast, concentrations of ATP, creatine, and PCr were similar in all muscles studied when expressed as ratios of [TCr]. Horse MGM had the lowest percent of water and protein per gram wet or dry weight but highest glycogen concentration of the muscles studied, irrespective of the reference base used to express concentrations. Coefficients of variation were lowest when muscle constituents were related to [TCr]. It is concluded that expressing muscle constituents relative to total creatine results in the smallest variation and is a good method for making comparisons between muscles of similar fiber composition. However, essential information concerning different types of muscle may be lost when this reference base is used.     

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.     

Changes in exopeptidase activities in skeletal muscles during disuse

Some aminopeptidase activities, dipeptidase-, tripeptidase-, and carboxypeptidase activities were measured in two different types of skeletal muscle in rabbit soleus muscle as a slow oxidative, and gastrocnemius muscle as a fast glycolytic type after immobilization in full extension with a plaster cast for 1, 2, 4, 7, 14 or 28 days. In correlation to the higher protein turnover in red muscles, the activities except of leucine and alanine aminopeptidase were higher in the normal soleus muscle than in the gastrocnemius muscle. Much higher activities of the tested enzymes were obtained in the immobilized soleus muscle than in the normal one after 2 weeks of immobilization. In the gastrocnemius muscle the tested enzyme activities generally did not change or decrease. The results demonstrate that the peptidases play a role in the process of protein breakdown in normal and disused skeletal muscles.     

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in "fast-twitch" muscle fibers but has little effect on enzyme activities in "slow-twitch" muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Morphological and biochemical changes in old rat muscles: effect of increased use

Male Wistar rats were strength and swim trained during a substantial period of old age to determine the influence of aging and activity on the histochemical and metabolic characteristics of a predominantly slow (soleus) and a predominantly fast (plantaris) skeletal muscle. Strength training counteracted the age-related atrophy of the fibers and the age-induced changes in fiber-type distribution of both muscles. Swim training, on the other hand, was without any effect on these parameters. The activity of both mitochondrial and cytoplasmic enzymes became lower with aging in the soleus muscle, whereas only the activity of the cytoplasmic enzymes became lower in the plantaris. Strength training reduced the aerobic capacity of both muscles, whereas swim training had the opposite effect. Aging induced a lower glycogen concentration of the lateral gastrocnemius muscle. This was avoided by swim training. The phosphocreatine and adenosine 5'-triphosphate concentrations were unchanged with aging but became higher with strength training. The activity pattern, therefore, seems to have a considerable influence on the age-related modification of the histochemical and metabolic characteristics of skeletal muscles of the rat. The effect, however, is related to the recruitment pattern of the fiber populations and the form of activity.