Simultaneous measurements of oxygen consumption and ammonia-N excretion in embryos and larvae of marine invertebrates.

The quantification of oxygen consumption and ammonia-N excretion rates is essential in determining energy requirements for development of larval invertebrates. In larval energetics, there is a need for accurate and uncomplicated techniques to quantify metabolic rates. A method for simultaneous measurements of oxygen and ammonia-N concentrations is presented. It employs sealed respirometric chambers (ca. 30 ml) in which embryos and larvae are incubated. Analysis is carried out in end-point samples by Winkler's titration and indophenol-blue for oxygen and ammonia-N, respectively. Water is sampled into volume-calibrated glass syringes and oxygen consumption and ammonia-N excretion rates were determined by the difference between experimental and control (no animals) units. The method was successfully used to measure metabolic rates in embryo and larval stages of the shrimp Farfantepenaeus paulensis and in veliger of the mussel Perna perna. The accuracy denoted by the coefficient of variation is comparable to previous results on larval metabolic rates. A biomass: volume (microg ml(-1)) is proposed to extend its application to further species of marine invertebrates. The method is simple to operate, involves non-expensive material and is portable enough for field work. A substantial number of replicates can be analyzed at the same time and O:N ratio, an indicator of the catabolized substrate, can be calculated.

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae): effects of development and temperature.

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.

Growth, oxygen consumption, ammonia-N excretion, biochemical composition and energy content of Farfantepenaeus paulensis Pérez-Farfante (Crustacea, Decapoda, Penaeidae) early postlarvae in different salinities.

Physiological responses of early postlarval shrimp Farfantepenaeus paulensis to different salinities were evaluated. Growth, net growth efficiency (K(2)), oxygen consumption, ammonia-N excretion, protein, lipid, carbohydrate, ash and energy content were determined in postlarval stages PL VI-VII and PL XIII-XV at 5 per thousand, 15 per thousand, 25 per thousand and 34 per thousand. PL VI-VII and PL XIII-XV correspond, in the wild, to the penetration of postlarvae into estuarine zones and the adoption of benthic habits, respectively. Postlarvae were gradually acclimated to the various salinity levels in the stage PL VI-VII and were maintained there until reaching the stage PL XIII-XV. Although development time was not affected by salinity, growth was decreased at 34 per thousand. Oxygen consumption was less affected by salinity while ammonia-N excretion presented a negative correlation with salinity. In both stages, protein content was lower at 34 per thousand. In PL VI-VII, lipid was decreased at 5 per thousand, while no significant difference in lipid content was observed in PL XIII-XV. Carbohydrate and ash levels did not vary in function of salinity levels. Energy content was reduced in PL XIII-XV at 34 per thousand. Net growth efficiency (K(2)) between PL VI-VII and PL XIII-XV presented higher values at 15 per thousand and 25 per thousand, and the lowest at 34 per thousand. Enhanced energy expenditure due to osmoregulation was not detected as denoted by oxygen consumption rates. Nitrogen metabolism seems to play a key role in maintaining body fluids concentration in relation to medium salinity. It is concluded that the high degree of euryhalinity in these stages of F. paulensis may be an adaptation to ontogenetic events such as migration to estuarine areas. For aquaculture purposes, it is recommended to maintain these postlarval stages at salinities between 15 per thousand and 25 per thousand rather than 34 per thousand.

A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells.

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.