Aerobic and anaerobic capacities differ in embryos of the annual killifish Austrofundulus limnaeus that develop on alternate developmental trajectories.

Embryos of the annual killifish Austrofundulus limnaeus have a remarkable tolerance to anoxia during their development, especially during diapause II (DII), but little is known about potential mechanisms by which this tolerance is achieved. This study examined the aerobic and anaerobic capacities of these embryos as they develop along alternate developmental trajectories and in response to altered incubation temperature. Aerobic and anaerobic capacities were estimated by measuring the total activity of the enzymes citrate synthase (CS) and lactate dehydrogenase (LDH), respectively. Embryos of A. limnaeus exhibit high anaerobic capacity throughout development as evidenced by high LDH/CS ratios, especially during early development through DII. Anaerobic production of lactate is supported by the heart isoform of LDH, even in stages of development that exhibit extreme tolerance of anoxia. CS capacity is extremely low during DII and may indicate an active suppression of mitochondrial metabolism during this stage of dormancy. Post-DII and "escape" embryos which bypass DII increase their aerobic and anaerobic capacities in tandem as they develop. The activity of both LDH and CS continue to increase for many days after morphological development ceases during DIII. Based on this observation, it is likely that regulation of metabolic dormancy is different in DII and III. Escape embryos seem to develop along a different metabolic trajectory than do embryos that enter diapause. Importantly, these embryos complete development with different enzymatic capacities that could influence physiological and ecological performance during early larval life.

Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers.

Diapausing embryos of Austrofundulus limnaeus survive desiccating conditions by reducing evaporative water loss. Over 40% of diapause II embryos survive 113 days of exposure to 75.5% relative humidity. An early loss of water from the perivitelline space occurs during days 1-2, but thereafter, rates of water loss are reduced to near zero. No dehydration of the embryonic tissue is indicated based on microscopic observations and the retention of bulk (freezable) water in embryos as judged by differential scanning calorimetry. Such high resistance to desiccation is unprecedented among aquatic vertebrates. Infrared spectroscopy indicates frequent intermolecular contacts via beta-sheet (14%) in hydrated egg envelopes (chorions). These beta-sheet contacts increase to 36% on dehydration of the egg envelope. Interestingly, the egg envelope is composed of protein fibrils with characteristics of amyloid fibrils usually associated with human disease. These features include a high proportion of intermolecular beta-sheet, positive staining and green birefringence with Congo red, and detection of long, unbranched fibrils with a diameter of 4-6 nm. The high resistance of diapause II embryos to water stress is not correlated with ontogenetic changes in the egg envelope.

Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus.

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via (14)CO(2)) into perchloric acid-precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole-embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.

Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles.

A previous phylogenetic analysis among 15 taxa of the teleost fish Fundulus suggested that there should be thermal-adaptive differences in heart metabolism among populations. To test this hypothesis, the rate of oxygen consumption and the activities of all 11 glycolytic enzymes were measured in isolated heart ventricle from two populations of Fundulus heteroclitus. Heart ventricular metabolism is greater in a northern population versus a southern population of these fish. Analysis of the amount of glycolytic enzymes indicates that 87% of the variation in cardiac metabolism within and between populations is explained by the variation in three enzymes (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase). These enzymes are the same three enzymes that were predicted to be important based on previously determined phylogenetic patterns of expression. Our data indicate that near-equilibrium enzymes, as well as classically defined rate-limiting enzymes, can also influence metabolism.

Anoxia tolerance of con-familial tiger beetle larvae is associated with differences in energy flow and anaerobiosis.

In this study, we compared survivorship, heat dissipation and biochemical features of anaerobiosis of two tiger beetle species (Coleoptera: Cicindelidae) exposed to anoxia. One species commonly experiences environmental immersion from rainfall and snowmelt (Cicindela togata), and the habitat of the other (Amblycheila cylindriformis) is not prone to flooding. The ancestral genus, A. cylindriformis, survives anoxia for only 2 days at 25 degrees C. In response to anoxia, these larvae immediately lose locomotory abilities, tissue concentrations of ATP fall precipitously within 12 h, and significant amounts of lactate are quickly produced. In contrast, C. togata larvae tolerate anoxia for 5 days. Heat dissipation is downregulated to a greater degree than that seen in A. cylindriformis (3.4% versus 14% of standard normoxic rate, respectively), the ability for locomotion is maintained and normoxic levels of ATP are defended for at least 24 h. Lactate is not accumulated until well into anoxic bout, and significant amounts of alanine are also produced. This study provides evidence that tiger beetles differ in physiological responses to anoxia, and that these differences are correlated with flooding risk and with species distribution.