The effects of water temperature, ionic content and total dissolved solids on Nephelopsis obscura and Erpobdella punctata (Hirudinoidea, Erpobdellidae)

Using a three-way factorial experimental design the effects of water temperature, ionic content (water type) and total dissolved solids on the mortality rates of Nephelopsis obscura and Erpobdella punctata were examined. A strong interaction between temperature and water type and temperature and total dissolved solids was observed on the mortality of E. punctata . Other than differences in mortality due to reproductive condition, N. obscura showed uniformly low mortality not significantly affected by temperature, water type or total dissolved solids. Thus the numerical dominance shown by N. obscura in Alberta and Western Canada could be explained by the differentially higher mortality of E. punctata with respect to the three tested independent variables.     

Comparative analysis of anoxic coleoptile elongation in rice varieties: relationship between coleoptile length and carbohydrate levels, fermentative metabolism and anaerobic gene expression

Rice ( Oryza sativa L.) seeds can germinate under anoxia and can show coleoptile elongation. The anoxic coleoptile is usually longer than aerobic coleoptiles. Although several hypotheses have been proposed to explain the ability of rice to elongate coleoptiles under anoxia, conclusive experimental evidence explaining this physiological trait is lacking. In order to investigate whether metabolic and molecular markers correlate with anoxic coleoptile length, we screened 141 Italian and 23 Sri Lankan rice cultivars for their ability to elongate coleoptiles under anoxia. Differences in anoxic coleoptile length were used to evaluate whether a correlation exists between coleoptile length and biochemical and molecular parameters. The expression of genes coding for glycolytic and fermentative enzymes showed a very low correlation with anoxic coleoptile length. Although differences were found in carbohydrate content between the varieties tested, this parameter also does not appear to be critical in terms of coleoptile elongation. Efficient ethanol fermentation does, however, correlate well with the elongation of coleoptiles under anoxic conditions.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

On the Physiological Role of Anaerobically Synthesized Lipids in <Emphasis Type="Italic">Oryza sativa</Emphasis> Seedlings

The objective of this work was to elucidate a possible adaptive role of lipid biosynthesis and unsaturated fatty acids (FAs), esterified to lipids, as terminal acceptors of electrons, alternative to molecular oxygen, in the shoots of rice seedlings (Oryza sativa L.) under conditions of strict anoxia. Biosynthesis of lipids and their accumulation, as well as the reduction of double bonds in unsaturated FAs, were studied by electron microscopic observation of the accumulation of lipid bodies in the cytoplasm and by the biochemical analysis of FAs in shoot lipids before and after anaerobic incubation of the shoots. The experiments were carried out with intact coleoptiles after 5 and 8 days of anaerobic germination of seeds (primary anoxia) and with detached shoots, preliminarily grown in air and then subjected to anoxia in the presence of 2% glucose for 48 h (secondary anoxia). In these experiments, lipid bodies did not accumulate in the cytoplasm under anoxic conditions. Lipid bodies appeared only during 48-h anaerobic incubation of detached coleoptiles in the absence of exogenous glucose, when mitochondria degraded. There was no change either in the double bond index of FAs, or in the qualitative and quantitative composition of FAs during shoot anaerobic incubation. We conclude that neither lipids synthesized under anaerobic conditions nor esterified unsaturated FAs are involved in plant adaptation to anaerobiosis as terminal acceptors of electrons, alternative to molecular oxygen. Lipid biosynthesis under anoxic conditions, which was demonstrated for anoxia-tolerant seedlings of Oryza sativa and Echinochloa phyllopogon in experiments with radioactive precursors, ¹⁴C-acetate and ³H-glycerol, is only the manifestation of a turnover of saturated FAs and various classes of lipids, which stabilizes cell membranes under adverse conditions of strict anoxia.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 540–548.Original Russian Text Copyright © 2005 by Generosova, Vartapetian.     

Respiration of Artemia franciscana embryos after continuous anoxia over 1-year period

Encysted embryos of the brine shrimp, Artemia franciscana, exhibit extraordinary longevity when exposed to continuous anoxia. To explore the metabolic basis of this ability, the post-anoxic respiration of embryos exposed to anoxia for periods exceeding 1 year was measured. Since anoxic metabolism might result in the accumulation of metabolic end products, an O₂ debt would be expected. Contrary to that expectation, post-anoxic embryos exhibited a marked depression in respiration rate whether embryos were hydrated under anoxic conditions or were exposed to a previous aerobic incubation and then placed under anoxia. These results, and those of previous studies, suggest that extended anoxia may bring the metabolism of these embryos to a reversible standstill.     

Anoxic injury accelerates phosphatidylcholine degradation in cultured cardiac myocytes by phospholipase C.

In neonatal cultured cardiac myocytes under normoxic conditions, 32Pi incorporation pattern into various phospholipids, and double-labeling experiments with 32Pi and [3H]methyl choline, suggest that phosphatidylcholine and phosphatidylinositol are turned over rapidly, whereas the turnover of phosphatidylethanolamine is probably much slower. While increased levels of the corresponding lysophospholipids were not found under anoxia, release of diacylglycerol and phosphorylcholine was observed. These data strongly suggest that phospholipase C, and not phospholipase A2, is involved in phospholipid degradation in cultured cardiomyocytes under anoxic conditions.     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.     

Effect of anoxia and hypoxia on brain lipid metabolism

Lipid metabolism in rat brain was investigated in mild hypoxia (5–7% O₂ in nitrogen), which is associated with no apparent change in energy metabolism, and in severe anoxic conditions (ischemic anoxia), which are associated with a rapid decrease in ATP and oxygen content in brain. When brain slices were incubated with labeled glucose or acetate, the amount of labeled CO₂ produced was no different in experimental and control conditions, but the incorporation of radioactivity into brain lipids was decreased in all hypoxic and anoxic conditions. Interestingly, the incorporation of label from [¹⁴C]glucose into phosphatidylinositols was specifically inhibited by both hypoxic conditions but not by conditions associated with anoxia. The incorporation of the same labeled precursor, i.e., [¹⁴C]glucose, into fatty acids was elevated in ischemic anoxia but reduced after mild hypoxia. Because of the obvious differences in oxygen utilization in brain in anoxic and hypoxic conditions, we believe that the observed disturbances in lipid metabolism may be due to factors other than those that arise from oxygen deficiency alone.     

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68–73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs. Handling editor: S. M. Thomaz     

Family-specific responses in survivorship and phenotypic traits to different light environments in a seedling population of Fagus crenata in a cool-temperate forest

Natural plant populations consist of individuals that exhibit variation in their phenotypic traits and demographic parameters. Here we report a study on maternal effects and the effects of different light environments on intra-specific variation in survivorship and ecologically relevant phenotypic traits of Fagus crenata seedlings in a cool-temperate forest community. We collected 901 seedlings from the ground beneath five maternal trees and used microsatellite DNA markers to identify maternal siblings that germinated naturally in the forest community. Selected seedlings were planted at three sites––one under a closed canopy with low light availability, one under a canopy gap in the natural forest community with moderate light availability, and one in a common garden with high light availability. The proportion of seedlings that were correctly assigned to their putative mother ranged from 60.0 to 82.7 % per maternal family, and 655 (72.7 %) seedlings in total were used for the analysis of survivorship and phenotypic traits. Among-family differences in survivorship remained after correcting for the effects of initial stem size during the first year after planting. However, this difference in survivorship became less pronounced in subsequent years. Seedlings grown under the canopy gap and/or in the common garden exhibited better performance in terms of phenotypic traits such as stem, leaf, and root morphology. In addition, seedlings of different maternal origins grown in the same environments had different individual leaf areas. These findings suggest that phenotypic variation due to maternal effects was a significant source of intra-specific variation within the local population.     

Growth of Daphnia longispina L. in a polyhumic lake under various availabilities of algal,bacterial and detrital food

The availability and importance of food sources for growth of Daphnia longispina L. from a highly coloured fishless lake with anoxic hypolimnion were assessed by combining in situ and laboratory experiments. In in situ experiments populations were enclosed in tubes with natural temperature stratification and with or without anoxic hypolimnion. In the laboratory experiments the importance of food source (littoral zone vs pelagic epilimnion) was assessed by enclosing moss thalli and a natural zooplankton population in a large-scale flow-through system supplying food for experimental Daphnia. Growth of juveniles of Daphnia in epilimnetic water was determined in batch culture experiments and the importance of increasing concentrations of bacteria and algae for their growth and development was investigated with a small-scale flow-through system. Access to the anoxic hypolimnion enhanced the growth of Daphnia by 23–24%. Growth rates in the tubes with anoxic hypolimnion were 0.36 and 0.16 d^–1 in July and August respectively. In tubes without anoxia the corresponding values were 0.29 and 0.13. In batch-cultures the highest growth rate determined was 0.16 and the overall rates were lower than in in situ experiments. In batch culture Daphnia was able to grow in darkness for 10 days with a rate of 0.16. In the large-scale flow-through system Daphnia population fed with littoral water reproduced well despite the low concentration of algae and increased its number by a factor of c. 32 in 10 days. However, the animals were small and net production of Daphnia population thus lower under the littoral influence than in the control treatment. Population could survive and grew slowly on pelagial water processed by a natural zooplankton community and with very little algae left. It is thus possible that bacteria serve as a life-support system enabling the population survival over periods of algal shortage. Small-scale flow-through experiments revealed that Daphnia longispina is able to mature and reproduce on a bacterial diet if the food concentration is high enough and Daphnia on bacterial food could achieve growth rates similar to those on an algal diet. The threshold food level for Daphnia longispina was estimated to be c. 18.5 g C 1^–1. Detrital material is of limited value in nutrition of Daphnia even in a lake where more than 75% of carbon is bound in particulate detritus.     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

Iron uptake of the normoxic, anoxic and postanoxic microglial cell line RAW 264.7

Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.     

CHO cell responses to low oxygen: regulation of oxygen consumption and sensitization to oxidative stress

We have investigated the regulation of oxygen consumption and modulation of glutathione levels in CHO-K1 cells under oxygen-limiting conditions. We report here suppression of oxygen consumption and alteration of the supply-dependent relationship as a consequence of prolonged hypoxic or anoxic exposure. The suppression is characterized by an increase in the value of P(o(2)/50) (the oxygen tension at which oxygen consumption is half maximal). Under prolonged anoxia there is also a decrease in the cells' potential to use oxygen. Elevated glucose consumption under low oxygen conditions may contribute to the suppression in respiration. The glutathione concentration remains constant throughout hypoxic exposure but may decrease by as much as 40% under anoxia. The glutathione level in hypoxic and anoxic cells increases by two- and four-fold, respectively, over that of the control cells when exposed to a cytotoxic level of oxygen (93%). This suggests that anoxic and hypoxic exposure sensitizes CHO cells to oxidative stress. (c) 1992 John Wiley & Sons, Inc.     

Anoxic or aerial survival of bivalves and other euryoxic invertebrates as a useful response to environmental stress—A comprehensive review

Laboratory and field studies have demonstrated the applicability of anoxic/aerial survival as an early warning indicator of contaminant induced stress. The effects of xenobiotics, including heavy metals, organometals and organics as well as contaminated field sediments have been investigated. The use of this physiological response is rapid, easily quantified and inexpensive. In most studies bivalve molluscs were investigated, the sea mussel Mytilus edulis L. being the favourite species.The different approaches used by various researchers, as reported in the literature, demonstrate the necessity for these experiments to be standardized. When estimating the mortality rate in a closed system or by aerial exposure, the cause of death may not be an indication of anoxia tolerance, but may be an artifact due to the development of microorganisms. Therefore the use of a flow-through system is suggested in cases where the duration of a test exceeds 1 week. When the response is used for the acute assessment of water quality a closed system may be used at low temperatures in combination with an organism with a relatively low anoxia tolerance (e.g., crustaceans).Various reasons why anoxic tolerance may be reduced by toxicants are proposed. Depletion of fuel reserves involved in anaerobic metabolism and intoxication by fermentative products may already commence during chronic exposure to contaminants, and/or the anaerobic energy metabolism may be disturbed. In general there is elevated catabolic activity during anoxic exposure, especially pyruvate derivatives are prominent end products in muscle tissue of contaminant exposed groups.comp biochem physiol 113C, 299–312, 1996.     

Regulation of anoxic death in Caenorhabditis elegans by mammalian apoptosis signal-regulating kinase (ASK) family proteins

Cells and organisms face anoxia in a wide variety of contexts, including ischemia and hibernation. Cells respond to anoxic conditions through multiple signaling pathways. We report that NSY-1, the Caenorhabditis elegans ortholog of mammalian apoptosis signal-regulating kinase (ASK) family of MAP kinase (MAPK) kinase kinases (MAP3Ks), regulates viability of animals in anoxia. Loss-of-function mutations of nsy-1 increased survival under anoxic conditions, and increased survival was also observed in animals with mutations in tir-1 and the MAPK kinase (MAP2K) sek-1, which are upstream and downstream factors of NSY-1, respectively. Consistent with these findings, anoxia was found to activate the p38 MAPK ortholog PMK-1, and this was suppressed in nsy-1 and tir-1 mutant animals. Furthermore, double-mutant analysis showed that the insulin-signaling pathway, which also regulates viability in anoxia, functioned in parallel to NSY-1. These results suggest that the TIR-1-NSY-1-SEK-1-PMK-1 pathway plays important roles in the reponse to anoxia in C. elegans.