Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp,Crangon crangon

Summary The mobilization of glycogen and phosphoarginine during work and their resynthesis during periods of recovery were investigated in abdominal muscles of the shrimpCrangon crangon. All parameters, metabolite levels as well as glycogen phosphorylase (EC 2.4.1.1) and synthase (EC 2.4.1.11) activities were determined in each individual shrimp investigated. At the onset of work both glycogen and phosphoarginine were degraded with the rate of phosphoarginine utilization being more than 80-fold faster than glycogen. After exhaustive work phosphoarginine stores were replenished within 30 min and seemed to exceed the resting level thereafter. In contrast, glycogen was not resynthesized immediately after work, but was further degraded during recovery leading to the accumulation of lactate. Only when the phosphagen level had reached the resting level did glycogenolysis shift to its resynthesis. The shift is characterized by: (1) a change in the mass action ratio of phosphoglucomutase from values below the equilibrium constant to values above the constant, (2) a dramatic decrease in the ratio fructose 1,6-bisphosphate/fructose 6-phosphate indicating phosphofructokinase inhibition, (3) an increase in the glucose concentration, and (4) an increase in the proportion of glycogen synthase I. The inactivation of glycogen phosphorylase by dephosphorylation during recovery was 2.4-fold. 36±8% (n=5) of total activity remained in the phosphorylated form. It is proposed that this part of the enzyme was inactivated by the drop in inorganic phosphate level due to the restoration of phosphoarginine.     

The influence of denervation of the maturation of the flight muscle of Periplaneta americana.

The metathoracic musculature of the American cockroach Periplaneta americana was denervated by dissecting the nerves originating in the metathoracic ganglion on one side within 2 days after the last moult. The biochemical and morphological differences between normal and denervated musculature were followed two weeks later. Denervation completely prevents the gain in weight, protein content and cytochrome oxidase activity which occur in normal muscles during the two weeks after previous moulting. The content of phosphoarginine in the denervated muscle does not differ from the control muscle and is lower than in young muscles. The ability to resynthesize phosphoarginine postanaerobically is substantially lower in the denervated muscle than in control muscles, or in young muscles immediately after the last moult. The ultramicroscopic structure of the denervated muscle differs from that of the normal muscle: the mitochondria possess less cristae, the matrix between them appears less dense and the sarcoplasmatic reticulum is less developed. Thus the denervated muscle not only fails to mature, but also developes serious structural and biochemical disorders.     

A new property of twitchin to restrict the “rolling” of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin-myosin interaction by “freezing” tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.     

Plasma glucocorticoid concentrations in free-ranging platypuses (Ornithorhynchus anatinus): response to capture and patterns in relation to reproduction

Energy demands in the platypus are likely to increase in the breeding season, which occurs from winter to early spring. Glucocorticoids, which play a major role in energy mobilisation, were measured in consecutive blood samples from free-ranging adults at approximately monthly intervals throughout the year. Glucose and free fatty acids were also measured in some samples. Plasma concentrations of glucocorticoids rose significantly during the first 30 min after capture, accompanied by a rise in free fatty acids, but no corresponding increase in glucose concentrations. We observed a strong pattern in plasma glucocorticoids in samples collected within 15 min of capture (indicative of pre-disturbance concentrations) in different phases of the annual reproductive cycle, with significantly higher levels in both males and females in the breeding season compared to the non-breeding season. These data, and the decline in tail fat stores that occur towards the end of the mating period (around October), suggest that platypus experience high-energy demands during this phase of reproduction. Plasma glucocorticoid concentrations in females sampled during the lactation period (October-February) were relatively low, and similar to those in females sampled in the non-breeding, non-lactation period (March-June). The latter requires further investigation as results may have been influenced by sampling limitations.     

Hemolymph cholinesterase activity in the Mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) that was exposed to adverse natural and anthropogenic conditions

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.     

Microbial activity and organic matter dynamics during 4 years of irrigation with treated wastewater

The global changes in rainfall frequency and quantity have subjected arid and semi-arid regions to long periods of drought. As this phenomenon corresponds to increasing trend of water shortage, the use of treated wastewater (TWW) has been suggested as an alternative for irrigation of agricultural crops in these areas. The aim of the study was to investigate the short- and middle-term effects of TWW irrigation on the soil microbial activities and organic carbon content. The microbial community activity was measured every 1–3 months for 4 years in a persimmon (Diospyros kaki) orchard. These activities were used here as an indicator for the soil health. The hydrolysis activity (detected by fluorescein diacetate hydrolysis (FDA) assay) increased during the irrigation season and was significantly higher in soils irrigated with TWW compared to those irrigated with freshwater (FW). This activity was also negatively correlated with dissolved organic carbon (DOC) concentrations during the irrigation season, suggesting that the community degraded the DOC in the soils regardless of its origin. The irrigation season was also characterized by an increase in nitrification potential in both TWW- and FW-irrigated soils, which coincided with high concentrations of nitrate (50 mg kg⁻¹ soil). Overall, there was an increase in all measured activities during the irrigation season, and they were higher in the TWW soils. However, it appears that after each irrigation season, the potential activity of the community returned to levels similar to or even slightly lower than those of FW-irrigated soil during the wet season, suggesting that the periodic irrigation did not significantly change the soil microbial activity.     

Development of the muscle system and contractile activity in the mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Mollusca,Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and fluorescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 μm in both mussel larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing video workstation. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult molluscs.     

Biosynthesis and role in osmoregulation of glycine-betaine in the Mediterranean mussel Mytilus galloprovincialis LMK

Quaternary bases, for example glycine-betaine, are difficult to quantify in biological materials because of a lack of specificity. However, nuclear magnetic resonance (NMR) can determine quaternary bases even in the presence of high water concentrations. Using NMR concentrations of glycine-betaine, the posterior adductor muscle of the Mediterranean mussel Mytilus galloprovincialis were measured up to 256 micromole/g dry weight. These concentrations were related to external salinity concentrations. The biosynthesis of glycine-betaine was demonstrated in M. galloprovincialis from the precursor (14)C choline.     

Control of glycolysis in the posterior adductor muscle of the sea musselMytilus edulis

Summary Concentrations of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate, phosphoarginine and citrate have been estimated after various periods of valve closure (Table 1 and Fig. 1). Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals glycogen phosphorylase, phosphofructokinase, hexosediphosphatase and pyruvate kinase catalyze non-equilibrium reactions. The first three enzymes possess relatively low activities (Table 2).From the changes in concentrations of the glycolytic intermediates it is concluded that phosphofructokinase controls the carbon flow during the first hours after valve closure, whereas later on the rate of conversion of phosphoenolpyruvate is determining this flow. In skeletal muscle phosphofructokinase controls the carbon flow during the whole period of exercise.The concentrations of ADP, AMP and inorganic phosphate increase, whereas the concentrations of ATP, phosphoarginine and citrate decrease during valve closure (Table 1 and Fig. 2). In contrast to skeletal muscle, these changes do not result in a strong increase in the glycolytic flux.There is a much greater potential for ATP hydrolysis by the myofibrillar ATPase system than is actually realized by the adductor muscle during valve closure.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

Anaerobic energy metabolism in isolated adductor muscle of the sea musselMytilus edulis L.

Summary Energy metabolism during anaerobiosis was investigated in the isolated posterior adductor muscle of the sea mussel. Metabolism appeared to be similar to that observed in the intact organism. Glycogen and aspartate are simultaneously utilized and levels of alanine, succinate, strombine and octopine increase. The sum of the adenylates remains constant, whereas phosphoarginine is dephosphorylated. The influence of iodoacetate, aminooxyacetate and hadacidin, inhibitors of glycolysis, transamination and purine nucleotide cycle, respectively, on the utilization of substrates and the interconversion of metabolites has been studied. The results suggest that the purine nucleotide cycle is not involved in the inverse correlation of changes in levels of aspartate and alanine, but that this exclusively depends on transamination reactions. Pyruvate (required for alanine formation) arises about equally from glycolysis and aspartate decarboxylation. When the utilization of aspartate is blocked by aminooxyacetate, glycolytically formed pyruvate is metabolized by reductive condensation with glycine and arginine to yield strombine and octopine. Under this condition phosphoarginine is dephosphorylated at a faster rate in order to maintain the energy status of the cell.Abbreviations Ac acetate - AEC Atkinson energy charge - Ala alanine - Asp aspartate - Glu glutamate - Lac lactate - Mal malate - Oct octopine - PA phosphoarginine - Prop propionate - Pyr pyruvate - Str strombine - Suc succinate     

31P NMR study of postmortem metabolism in porcine and bovine muscles

31P NMR spectroscopy was used to evaluate interspecies differences in muscle fibre types and related postmortem metabolism. M. longissimus thoracis (MLT) and m. pectoralis superficialis (MPS) of bulls and MLT of pigs were investigated. In perchloric acid extracts NMR resonances for sugar phosphates (SP), inorganic phosphate (Pi), glycerophosphorylcholine (GPC), phosphocreatine (PCr), adenosine triposphate (ATP), adenosine diphosphate (ADP) as well as for NAD+/NADH could be distinguished. Also, glycogen and lactate contents and pH were determined. The relative contents of phosphorus compounds in bovine muscles of similar participation of muscle fibre are similar. Bovine muscles contain a relatively large proportion of PCr (48% of all phosphates 15 minutes post-mortem in MPS) whereas porcine MLT show lower PCr content (11% 15 minutes post-mortem). On the other hand, the ATP content is relatively higher in porcine MLT when compared with bovine muscles in the early phases of the postmortem processes. No NMR-detectable levels of GPC were measured in porcine MLT in contrast to bovine muscles. This suggests that the GPC content does not depend solely on the fibre participation but is also animal species determined. The 24 hour postmortem metabolism patterns of bovine and porcine muscles have many common traits. CP disappeared first followed by ATP. Simultaneously, the Pi concentrations increased. However, the content of SP remained relatively constant in porcine, but not in bovine muscles where it increased only gradually. The significantly higher concentrations of SP and lactate as well as the lower values of glycogen and pH measured for porcine as compared with bovine muscles suggest an enhanced glycolysis during the early phases of postmortem processes in porcine muscles.     

Seasonal changes in the concentrations of plasma gonadotropins and prolactin in wild mallard drakes

Seasonal changes in the concentrations of plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin were measured in serial samples taken from seven captive wild mallard drakes exposed to natural lighting and temperature in Kiel, West Germany (54 degrees N), for 20 months. The seasonal pattern of plasma LH levels was characterized by high titers during the reproductive phase in the spring, a steep decrease toward the end of this phase (May/June), low levels during the summer, and a second annual peak in the fall. Plasma FSH levels increased during February and March, the period of rapid testicular growth, and reached the highest values at the end of March/beginning of April. Later in the spring FSH levels decreased and remained low for the rest of the year. The concentrations of plasma prolactin increased progressively during April and May, reaching their highest values at the end of the breeding season, coinciding with the steep fall in the levels of plasma gonadotropins. Prolactin concentrations fell during July and August and were at their lowest level in the autumn. It is concluded that the development of photorefractoriness is associated with an increase in the concentrations of plasma prolactin.     

An analysis of the pollution problem in Slavonski Brod (eastern Croatia)

H2S, PM2.5, O3, NO2, SO2 and meteorological parameters such as temperature, relative humidity, precipitation, wind speed and wind direction were measured simultaneously in an eastern Croatian town called Slavonski Brod during the season winter/spring 2010. Emissions from the nearby cross-border (Bosnia and Herzegovina) oil refinery were identified as sources of temporary elevated concentrations of H2S. The maximum daily averages of PM2.5 concentrations during the winter period were as high as 240 microg m(-3) which is a value 10 times greater than the threshold prescribed by the World Health Organization. It is considered that the heating season, dense traffic, intense industrial activities and temperature inversion during stable weather conditions are prevailing contributors to higher winter concentrations of PM2.5. The results of the principal component analysis technique (PCA) have shown that lower air temperature, lower wind speed and higher relative humidity play a significant role in the winter pollution episodes. From a public health point of view, implementation of measures aimed at reducing the levels of H2S and PM2.5 should be considered.     

Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.     

Age at sexual maturity of males and timing of the mating season of polar bears (Ursus maritimus) in Greenland

Age at sexual maturity among male polar bears (Ursus maritimus) and timing of the mating season were studied by microscopic and macroscopic analysis of the reproductive organs from 165 male and 92 female Greenland polar bears. Males became sexually mature in the spring following their fifth birthday. Testes sampled during the spring reached 95% of their asymptotic length at 5.8 years. Some spermatozoa were found in 2- to 4-year-old bears during the spring, but only in low concentrations in a mixture of other cell types. The first corpus luteum was found on 1 April and most ovulations were estimated to occur before mid-May which indicate that the peak of the mating season begins in late March and ends in May. These findings are somewhat different from a similar study from Svalbard, where the mating season is estimated to peak later and male polar bears are found to become mature earlier. We hypothesize that these differences occur because adolescent males have their peak testicular activity after the main breeding period and therefore appear to be fully sexually mature during summer and not during spring.     

Rearing management of rabbit males selected by high growth rate: the effect of diet and season on semen characteristics

A total of sixty-six young males were used to evaluate the effect of low (L), medium (M) and high (H) concentrations of dietary digestible energy received during the rearing seasons (autumn and spring) on the performance and main semen characteristics of males for artificial insemination selected by a high growth rate. Males reared during the spring season presented a significantly higher weight at weaning than those reared during the autumn season (P < 0.001), and these differences were maintained until the end of the trial. The requirements of the males were easily covered as a general rule. In the autumn group, the males were unable to intake the digestible protein recommended only during their 3rd month of life, especially with low concentrate diets (P < 0.05). H males showed higher semen concentration and production during the autumn season, while L males showed a higher semen concentration and production than M males during the spring season, the H group showed intermediate values (P < 0.001). Males reared during the spring season showed significantly higher values of sperm concentration (P < 0.01) and production (P < 0.01). H males presented a lower percentage of spermatozoa with cytoplasmic droplets than the L group (P < 0.05) and the lowest values for sperm abnormalities during the autumn season, while the L group presented higher values for percentage abnormalities, especially during the last month controlled (P < 0.05). As a general rule, the main motility parameters controlled were not affected by the rearing diet received nor the season. These results seem to indicate that the management of rabbit males during the growing and rearing periods seem to significantly affect their subsequent performance and semen production.     

The first decade of oligotrophication in Lake Constance

Phytoplankton biomass and species composition were measured with a relatively high temporal resolution (once or twice a week during the growing season) from 1979 to 1989 in Lake Constance/Überlingersee. Over this period soluble reactive phosphorus (SRP) concentrations during winter mixing were reduced by ca. 50% from 104 to 47 g 1^–1, which caused a prolongation and amplification of the epilimnetic P depletion during the growth period. Seasonal dynamics of phytoplankton reacted to the decrease of SRP in the following ways: (1) Algal biomass decreased at least proportionally to the winter SRP concentrations in summer, but not in spring and autumn when biomass fluctuated irregularly. (2) The peak of biomass concentration changed from summer to spring. (3) The earlier onset of epilimnetic P depletion during the season in recent years promoted a stronger growth of some pennate diatoms in spring. It caused an amplification of the silicon depletion in summer, which may cause still greater reduction of diatoms and total algal biomass in summer. (4) Reduction of algal biomass during the clear-water phase proper became shorter and less pronounced. (5) The temporal variability of algal biomass decreased in summer and autumn but not in spring. (6) Average cell sizes remained unchanged in summer and autumn but increased in spring during the beginning of oligotrophication. These results are largely in agreement with other studies on lake restoration and expectations derived from the PEG (Plankton Ecology Group) model (Sommer et al. 1986). They show that a 50% reduction of SRP concentrations during homothermy may have pronounced effects on seasonal dynamics of algal biomass in a large and deep lake. The algal response to the external change of SRP concentrations can be described by the Le Chatelier principle, implying that the internal structure of the system (e.g. species composition) changes in order to minimize the effect of the external pressure (e.g. reduction of total biomass). Suggestions are made as to how this system behaviour may emerge from local interactions.     

Seasonal variations in the rates of aquatic and aerial respiration and ammonium excretion of the ribbed mussel, Geukensia demissa (Dillwyn)

Aquatic and aerial rates of oxygen consumption and ammonium excretion of ribbed mussels, Geukensia demissa (Dillwyn), collected from the mid-intertidal zone of a mid-Atlantic salt marsh, were measured under ambient conditions of food, temperature, and salinity over five seasons. Rates of aquatic respiration covaried with body size and season, as the rates were high and strongly related to mussel tissue weight in spring and summer but low and weight independent in winter. There was a significant interannual difference between summer of 1995 and 1996. Rates of aerial respiration also varied seasonally, with high rates of oxygen consumption in summer and low rates in winter. The magnitude of these seasonal variations were greater than those for aquatic respiration, and as a result, the ratio of aerial to aquatic respiration was higher in summer (0.61 and 0.52) than in winter (0.11). This indicates that G. demissa was able to actively regulate aerial respiration, thereby permitting high aerobic metabolism during prolonged periods of air exposure in summer. We hypothesize that such high rates of aerial respiration, during the seasons of high metabolic activity, are required to provide sufficient energy for mussels to facilitate food digestion during air exposure at low tide. Rates of ammonium excretion varied seasonally and increased with mussel weight in all seasons. The atomic ratio of oxygen to nitrogen (O:N), calculated from aquatic respiration vs. ammonium excretion, was significantly lower in autumn (26) than in other seasons (46–60) among which the O:N did not vary significantly.