Anaerobic metabolism in aquatic insect larvae: studies onChironomus thummi andCulex pipiens

Summary The metabolic effect of hypoxia and anoxia on the larvae ofChironomus thummi andCulex pipiens was investigated. InC. thummi anoxia resulted in a characteristic decrease of ATP and P-arginine concentrations and in an accumulation of alanine and lactate within 60 minutes. These changes continued during prolonged incubation but at lower rates. Ethanol, the major product during long-term anoxia, was largely excreted into the ambient water.A significant accumulation of these metabolites occurred only at a of 7 Torr. However, the proportion of anaerobic energy production even at this low amounted to less than 5% of the total energy consumption measured during experimental anoxia. Thus the chironomid larvae exhibited a remarkable capacity for utilizing very low levels of oxygen to maintain an aerobic metabolism. Complete anaerobiosis was observed only under anoxic conditions.Recovery from prior anoxia began with the reestablishment of normal ATP, P-arginine and succinate concentrations, whereas removal of the accumulated alanine and lactate and replenishment of the normally high level of malate required several hours. Culex larvae were shown to have a very low anaerobic capacity and a high rate of lactate accumulation.The significance of the results is discussed with particular emphasis on comparative aspects.     

Pyruvate metabolism in rice coleoptiles under anaerobiosis

Relative importance of ethanolic, lactate and alanine fermentation pathways was estimated in coleoptiles of rice seedlings (Oryza sativa L.) subjected to anoxic stress. The in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and alanine aminotransferase (AlaAT, EC 2.6.1.2) in the coleoptiles increased in anoxia, whereas no significant increase was measured in lactate dehydrogenase (LDH, EC 1.1.1.27) activity. At 48 h, the ADH, PDC and AlaAT activities in anoxic coleoptiles were 62-, 15- and 7.6-fold greater, respectively, than those in the presence of oxygen. Ethanol and alanine in the coleoptiles accumulated rapidly under anoxia, increasing by 48 h, 57- and 5.6-fold compared with those in the presence of oxygen, respectively. However, lactate concentration did not increase and no initial burst of lactate production was detected. The relative ratio of carbon flux from pyruvate to ethanol, lactate and alanine in anoxic coleoptiles was estimated to be 92, 1 and 7% of the total carbon flux, respectively. These results suggest that the potential carbon flux from pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and alanine in rice coleoptiles during anoxia.     

Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance

The efficiency of ethanolic fermentation in anoxia tolerance under sugar-limiting conditions, as in the field is still matter of debate. Due to higher rates of glycolysis and ethanol fermentation, faster depletion of sugar stores leads to decreased survival. In the present work the hypothesis that alanine amino transferase ( AlaAT ) fermentation be involved in anoxia tolerance was explored in Medicago truncatula during germination and seedling establishment. Expression of AlaAT and two low oxygen-responsive genes, alcohol dehydrogenase ( ADH ) and lactate dehydrogenase ( LDH ) were determined by real time quantitative RT-PCR and AlaAT activity was determined by ¹⁵N-Glutamate labelling coupled to amino acids analysis by gas chromatography–mass spectrometry and HPLC. Under anoxia not only ADH and LDH levels of expression increased but also AlaAT expression increased substantially. In parallel in vivo AlaAT activity increased and resulted in an increase in alanine synthesis that accumulated as the major amino acid instead of asparigine. These findings support the hypothesis that AlaAT expression and alanine accumulation contribute efficiently to anoxia tolerance. By competing with ethanolic fermentation for pyruvate, under sugar-limiting conditions alanine synthesis saves C3 skeletons avoiding a shortage in carbon availability and limits accumulation of acetaldehyde, a toxic compound. On another hand, increase in alanine was accompanied by an increase in γ-amino butyric acid, both amino acids may intervene in cytosolic pH regulation. Finally the role of alanine in anoxia tolerance was strengthened by the fact that when alanine synthesis was impaired germination and seedling development failed under anoxia.     

The effect of oscillating dissolved oxygen concentrations on the metabolism of a Spodoptera frugiperda IPLB-Sf21-AE clonal isolate

The effect of oscillating dissolved oxygen (DO) concentration on the metabolism of a clonal isolate of the Spodoptera frugiperda IPLB-Sf21-AE insect cell line was investigated. Specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. Prior to conducting the oscillating DO experiments, it was found that the DO concentration could be reduced to 15% air saturation without adversely affecting the growth rate. Under these conditions, glucose and glutamine became depleted as the maximum cell density was reached. The introduction of DO oscillations, that is, cycles consisting of 30 min at 15% DO followed by 30 min of anoxia, significantly altered cell metabolism, including inhibition of cell growth and recombinant protein synthesis. The effect of DO oscillations on glucose consumption was dependent on the experimental conditions. Glucose exhaustion occurred when the DO oscillations contained either an "apparent" anoxia period (nitrogen sparging discontinued upon reaching 0% DO) without pH control or a "true" anoxia period (nitrogen sparging continued throughout anoxia period) with pH control. Glucose consumption was significantly decreased, however, when the cells were exposed to a "true" anoxia period without pH control, that is, low pH inhibited glucose utilization. Glutamine uptake was not significantly affected by DO oscillations. Lactate only accumulated in the oscillating DO runs, a finding consistent with previous results demonstrating that significant lactate accumulation only occurs under DO-limited conditions. (c) 1995 John Wiley & Sons, Inc.     

Winter survival of an adult bark beetle Ips acuminatus Gyll

Freezing-susceptible adult Ips acuminatus hibernate underneath bark of Scots pine. The beetles lower their supercooling points from ?20 to ?34°C due to accumulation of low molecular weight antifreezes. The capability of specimens to supercool to about ?20°C in the absence of cryoprotective solutes during winter, seemed to be at least partially attributable to the presence of a thermal hysteresis factor at 3–4°C.Using a GC-MS-COM technique, a unique combination of accumulated solutes present only in specimens demonstrating supercooling points below ?20°C was identified as ethylene glycol, mannitol, sorbitol and dulcitol. Not previously found in nature, ethylene glycol was the major solute (90%) synthesized at sub-zero temperatures. Exposure to ?10°C was an effective cue to accumulation of ethylene glycol and nearly 5 times as effective in promoting sorbitol synthesis than was ?5°C. When low molecular weight substances were lost at high temperatures, they were not re-synthesized in beetles re-exposed to sub-zero temperature. The supercooling point was closely related to both the concentration of ethylene glycol and to the haemolymph melting point. Attempts to correlate changes in sorbitol concentrations to changes in supercooling points were not conclusive.Proliferation of thermal hysteresis was observed in the beginning of November. A melting-hysteresis freezing point differential of about 3.6°C was demonstrated in the haemolymph of beetles during December. No thermal hysteresis was demonstrated in the haemolymph of positive phototactic beetles or in the outdoor beetles in May. The combination of high temperature and long photoperiod appeared to be a more effective cue to the final loss of thermal hysteresis than was high temperature alone.     

Respiratory metabolism in buckwheat seedlings

Young seedlings of buckwheat (Fagopyrum esculentum) respire in air with an RQ of unity. Analysis of respiratory substrates coupled with a study of the utilization of acetate-¹⁴C and glucose-¹⁴C suggest that both the Embden-Meyerhof-Parnas, tricarboxylic acid and pentose phosphate sequences participate in the total respiratory catabolism.

In anoxia CO₂ dropped to one third of the aerobic rate and ethanol accumulated to only about one half the rate of CO₂ output on a molar basis. Smaller amounts of lactate, succinate and free amino acids (particularly alanine and γ-aminobutyric acid) accumulated, carboxylic acids decreased and there were initial increased in pyruvate and α-ketoglutarate. The observed changes are consistent with residual tricarboxylic acid and pentose phosphate cycle activity in anoxia and may account for the excess CO₂ production over ethanol accumulation. CO₂, ethanol and lactate production did not account for all of the carbohydrate consumed in anoxia.

Relative rates of carbon loss were measured in air and in atmospheres containing 3.5%, 2.1%, 1.3% and 0.6% oxygen. The extinction point of anaerobic metabolism was 1.5%.

On return to air from anoxia the CO₂ output increased and the RQ rose from 0.8 to 1.0 over the first 2-hour period. Ethanol, lactate and succinate were consumed and other constituents returned to their previous aerobic level. Some of these changes suggest a rather slow resumption of tricarboxylic acid cycle activity on return to air.

Carbon loss as CO₂ in air was greater than the carbon loss as CO₂ at the extinction point. Carbon loss in anoxia as CO₂, ethanol and lactate was similar to carbon loss at the extinction point. Assessed in this orthodox manner buckwheat seedlings show no Pasteur effect but the complex nature of the changes in levels of metabolic substrates and intermediates do not allow firm conclusions to be drawn on the effects of oxygen on the rates of glycolysis and other respiratory processes.

     

Energy metabolism and metabolic rate of the alder leaf beetle Agelastica alni (L.) (Coleoptera,Chrysomelidae) under aerobic and anaerobic conditions: a microcalorimetric study

In early fall, adult alder leaf beetles (Agelastica alni L.) retreat, for overwintering, to the top layer of the soil near their forage trees where the ground gets easily waterlogged so that the beetles will be submerged and cut off from atmospheric oxygen. Hence, unlike most other adult insects, alder leaf beetles encounter hypoxia/anoxia in their natural habitat and this may occur at moderate temperature. Exposing beetles to pure nitrogen gas at 20 degrees C had similar behavioral and metabolic effects as submerging them in water, causing rapid immobility and increasing the content of lactate about sevenfold to some 5&mgr;molg(-1) body weight during 10h anoxia. Recovery from 10 h hypoxia/anoxia in pure nitrogen was complete within about 90min.Hypoxia/anoxia triggered a marked decrease in metabolic activity in the beetles (microcalorimetry at 21.7 degrees C) as indicated by a precipitous drop in their heat flow rate, from 1.39+/-0.27 to 0.08+/-0.04mWg(-1) body weight, i.e. by about 94%, when the flow of gas through the calorimeter was switched from air to pure nitrogen. Post-anoxic recovery was accompanied by a peak in heat flow rate that exceeded the basal normoxic rate by about 50%. The homoeostasis of adenine nucleotides in Agelastica is lost when oxygen is wanting. Submergence at 15 degrees C for three days caused a dramatic fall in ATP, to less than 2% of the normoxic value, and a marked increase in AMP, while the total contents of adenine nucleotides decreased by almost two-thirds. Reduced metabolic activity, combined with the capacity to regenerate ATP after readmission of air, is regarded as a key factor for surviving transient lack of oxygen in alder leaf beetles.     

Decay of cytochromes and appearance of a cyanide-insensitive electron transfer pathway in Euglena gracilis grown in anoxia

The effect of anoxia over a 3-week period on the respiratory ability of Euglena gracilis (strains Z and ZC) was studied. Low temperature absorption spectra indicated that comparable alterations of the cytochromes occurred in both cell types studies, leading to the disappearance of cytochrome oxidase and cytochrome c558 in the aplastic strain ZC and in the wild type strain Z. Both types of cells maintained their ability to consume O₂ when they were transferred from the nitrogen atmosphere to air. Resistance of respiration to cyanide and azide, and sensitivity to propyl gallate increased during anoxia, indicating the decreasing role of cytochrome oxidase in this O₂ consumption. Quantitative changes in the O₂ consumption capacity were followed during the course of anoxia; this capacity decreased during the 5 first days of anoxia and then increased to recover its initial value during the second week. This recovery of a high O₂, consumption capacity was linked to the appearance of a cyanide- and azide-resistant, propyl gallate-sensitive O₂ consumption pathway. This paper raises the question of the physiological significance of this electron transfer pathway induced by anoxia.     

Stimulation of glycolysis in anaerobic elongation of pondweed (Potamogeton distinctus) turions

Stem segments prepared from pondweed (Potamo geton distinctus A. Benn.) turions (overwintering buds) elongate in anaerobic conditions, whereas there is almost no elongation in air. The anaerobic elongation was accompanied by a decrease in dry weights of stem segments, mainly due to consumption of storage starch in the amyloplasts of stem cells. On the other hand, total contents of amino acids increased in stem segments, in which contents of alanine, valine, leucine, and isoleucine increased, but contents of asparatic acid decreased. Moreover, contents of lactate in stem tissues increased at an early stage of anaerobic incubation. In tracer experiments with 14C-glucose, 14C incorporation into stem tissues in anoxia was only half of that in normoxia. However, conversion of 14C to ethanol occurred exclusively in anoxia. 14C-labelled metabolites were analysed by two-dimensional cellulose thin-layer chromatography. 14C incorporation into sucrose and alanine was significantly increased in anoxia. The activity of alanine aminotransferase was enhanced by anoxia, suggesting that pyruvate is a precursor of alanine synthesis. The results suggest that pondweed turions produce energy necessary for anaerobic elongation by activating conversion of storage starch in the amyloplasts to ethanol, alanine and lactate.     

Tissue specificity of metabolism in the bivalve mollusc <Emphasis Type="Italic">Anadara inaequivalvis</Emphasis> Br. under conditions of experimental anoxia

Peculiarities of the course of metabolic processes in tissues of the bivalve mollusc Anadara inaequivalvis Br. were studied under conditions of experimental anoxia. In the absence of oxygen, in gill and foot the protein catabolism processes were found to be enhanced; this led to a decrease of the protein content and to an increase of the free amino acid and urea levels. Predominantly hydrolyzed were low molecular peptides, which was indicated by a decrease of the cathepsin D activity on the background of a rise of the γ-glutamyltranspeptidase activity. Anoxia was accompanied by enhancement of the succinate thiokinase and fumarate reductase reactions controlled by alanine and aspartate aminotransferases. This prevented accumulation of toxic lactate in tissues and allowed obtaining an additional macroerg resource. Metabolic processes in the mollusc hepatopancreas were oriented to production of amino acids.     

Effects of anaerobiosis on carbohydrate oxidation by roots of Pisum sativum

The aim of this work was to discover the effects of anaerobiosis on the breakdown of sugars by the apical 6 mm of the roots of 5-day-old seedlings of Pisum sativum. Estimates of the maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase and NADP-specific malic enzyme showed them to be comparable to that of phosphofructokinase. Metabolism of sucrose-[U-¹⁴C] by excised apices was restricted by anoxia mainly to conversion to ethanol, CO₂ alanine and glycolytic intermediates. Measurements of metabolites over a period of 240 min after transfer of excised apices to nitrogen showed a marked and continual accumulation of ethanol, a smaller continual accumulation of alanine, a small initial rise in lactate and no detectable accumulation of malate or pyruvate. The rates of CO₂ production, of accumulation of ethanol and alanine, and of the labelling of these compounds by sucrose-[¹⁴C] declined markedly during the first 240 min of anaerobiosis. The conclusion is that under anaerobic conditions carbohydrate metabolism in the pea root apex is largely restricted to alcoholic fermentation, and, to a lesser degree, to alanine production.     

Lactic Acid efflux as a mechanism of hypoxic acclimation of maize root tips to anoxia

Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.     

Evaluation of the importance of lactate for the activation of ethanolic fermentation in lettuce roots in anoxia

According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of lactate production and the resulting pH drop in the cytoplasm activates ethanolic fermentation. To evaluate this system in lettuce ( Lactuca sativa L.), seedlings were subjected to anoxia and in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) and concentrations of ethanol, acetaldehyde and lactate were determined in roots of the seedlings. The in vitro activities of ADH and PDC in the roots increase in anoxia, whereas no significant increase was measured in LDH activity. At 6 h, the ADH and PDC activities in the roots kept in anoxia were 2.8- and 2.9-fold greater than those in air, respectively. Ethanol and acetaldehyde in the roots accumulated rapidly in anoxia and increased 8- and 4-fold compared with those in air by 6 h, respectively. However, lactate concentration did not increase and an initial burst of lactate production was not found. Thus, ethanol and acetaldehyde production occurred without an increase in lactate synthesis. Treatments with antimycin A and salicylhydroxamic acid, which are respiratory inhibitors, to the lettuce seedlings in the presence of oxygen increased the concentrations of ethanol and acetaldehyde but not of lactate. These results suggest that ethanolic fermentation may be activated without preceding activation of lactate fermentation and may be not regulated by oxygen concentration directly.     

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 °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. Accepted: 1 March 2000     

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.     

The Role of Sugars,Hexokinase, and Sucrose Synthase in the Determination of Hypoxically Induced Tolerance to Anoxia in Tomato Roots

Hypoxic pretreatment of tomato (Lycopersicon esculentum M.) roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if external sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Several enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.     

OBSERVATIONS ON THE RESPIRATION OF TRYPANOSOMA CRUZI IN CULTURE

1. The oxygen consumption of cultural forms of Trypanosoma cruzi decreases in intensity with increasing age of the cultures; no correlation with any other factor studied could be established. 2. The respiratory quotient was high for the first 10 days, i.e. as long as the population increased; with the onset of a decline in numbers, the R.Q. began to drop. It is believed that the flagellates consume in the beginning predominantly sugar and later predominantly protein. Observations on the pH of the cultures bear out this view. 3. The oxygen consumption was independent of the oxygen tension over a wide range of tensions. 4. The oxygen consumption increased in the temperature range 13° to 40°C., while a temperature of 44°C. proved to be lethal. Upon application of Arrhenius'' equation, two straight lines, intersecting at about 28°C., resulted. The µ values were 23,980 and 5275 for the lower and higher temperature range respectively. 5. Of the oxidase inhibitors tested, strong inhibition of the oxygen consumption was achieved with azide, cyanide, and hydrogen sulfide. Pyrophosphate had no influence at all. There is some probability that cytochrome oxidase is the chief oxidase present. 6. The strongest inhibitory influence due to dehydrogenase inhibitors was observed with propyl carbamate and high concentrations of ethyl carbamate. 7. A small fraction of the oxygen consumption, about 10 per cent, may be due to substances with sulfhydryl groups, as indicated by a slight but distinct inhibition due to dilute iodoacetate and to arsenite.     

Production and organ distribution of succinate in rice seedlings during anoxia

Anaerobic production of succinate, a common feature in animals able to sustain anoxia, has seldom been reported in plants. By the use of ¹H-nuclear magnetic resonance spectroscopy we show here that succinate is produced by rice seedlings (Oryza sativa L. cv. Arborio) subjected to anoxic conditions. Starting from levels below I μmol (g fresh weight)⁻¹ in air, after 48 h of anoxia the levels of alanine, succinate and lactate had increased to 23.8, 5.2 and 1.0 μmol (g fresh weight) ⁻¹, respectively, in shoot tissues. Succinate was accumulated in shoots, notably in the coleoptiles, but not in roots of the rice seedlings, suggesting its involvement in rice coleoptile elongation under anoxia. Other possible functions of succinate production in rice seedling, an organism highly tolerant to anoxia, are discussed.