Near anoxia and sulfide as possible factors influencing the spatial distribution of Acartia tonsa and Acartia clausi: Comparative evaluation of egg tolerance

In many coastal and estuarine areas the planktonic copepods Acartia tonsa and Acartia clausi show a spatial separation with A. tonsa restricted to brackish waters and confined environments and A. clausi inhabiting areas more influenced by sea water. The hatching and viability of A. tonsa and A. clausi eggs exposed to anoxia and anoxia/sulfide (conditions that are frequent in bottom waters of the most confined areas) was evaluated to determine if these stress factors play a role in the distribution of these species. Subitaneous eggs, spawned by laboratory reared organisms, were incubated in near anoxia (< 7.59 × 10^− 3 mmol O₂ L^− 1) or anoxia/sulfide (∼ 1 mmol L^− 1) for different periods (1, 4, 8 and 15 days), then transferred to normoxic conditions. The exposure of the eggs to near anoxia and sulfide appears to induce the same response (quiescence) in both species. Exposure times ≤ 8 days to near anoxia or anoxia/sulfide did not affect egg viability, while 15 day exposure caused significant declines in hatching success of both species. A significant difference between the effects of near anoxia and anoxia/sulfide was observed when incubation lasted 15 days; hatching of eggs exposed to sulfide being higher than that of eggs exposed to near anoxia for both species. No significant differences were observed between the two species in hatching success of eggs exposed to both near anoxia and anoxia/sulfide (with the exception of eggs incubated in near anoxia for 4 days). The results indicate that the impact of anoxia and sulfide on the eggs of the two Acartia species cannot be a factor explaining the spatial distribution in coastal and brackish environments of these copepods.Feeding experiments on A. clausi were also performed. Suitability of different algal species to rear this copepod was evaluated and the results were compared with data previously obtained for A. tonsa. Differences in feeding needs between A. clausi and A. tonsa are discussed.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

NO- and H2S brain systems of the Japanese shore crab Hemigrapsus sanguineus under conditions of anoxia

The topography and dynamics of the activity of the enzymes of the synthesis of nitric oxide (NO) and hydrogen sulfide (H₂S) in the brain of the shore crab Hemigrapsus sanguineus after 1, 6, and 12 h of anoxia was studied histochemically and immunocytochemically. Changes in the activity and number of NO- and CBS-immune-positive cells that take place due to anoxia and the intensity of which depends on the duration of the influence were revealed. The fact that the balance between the nitric oxide and hydrogen sulfide systems in the brain of the crabs H. sanguineus is preserved indicates the joint participation of those systems in the central regulation of adaptive mechanisms under the influence of anoxia and, apparently, plays an important role in the adaptation of these hydrobionts to oxygen deficit.     

Fluorometric measurement of pyridine nucleotide reduction in the giant axon of the squid

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1–2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25–50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10–20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon.     

Effects of filamentous algal mats on sulfide invasion in eelgrass (Zostera marina)

The effect of filamentous algae invasion into Zostera marina meadows on water quality, sediment sulfur pools and sulfide invasion into plant tissues was studied experimentally. Sulfide invasion was assessed through analysis of sulfur isotopic composition (δ³⁴S) and total sulfur (TS) concentrations in plant tissues. The algal mats (5 and 10 cm thickness) depleted oxygen in the mats and increased the pools of sulfides in the sediments. Plants exposed to algal mats had δ³⁴S signals closer to the δ³⁴S of sediment sulfide, whereas plants with no mats present had δ³⁴S signals closer to the δ³⁴S of seawater sulfate, indicating a higher sulfide invasion in plants exposed to algal mats. The δ³⁴S varied between the plant tissues with the leaves having more positive δ³⁴S signals than roots and rhizomes, indicating that sulfide was invading into the roots and moved to the other tissues through the lacunae. TS concentrations were higher in plants exposed to algal mats suggesting that sulfur derived from sediment sulfide accumulated in the plants. F_sulfide showed that up to 50% of the sulfides in the plants were derived from sedimentary sulfides. The combined effect of water column anoxia in the lower parts of the meadow and high sulfide invasion into the plants lead to significantly reduced growth rates after 3 weeks and the below-ground tissues showed signs of degradation suggesting that algal mats invasion in to Zostera marina meadows can result in seagrass decline.     

The reaction of clostridial ferredoxin with cyanide

Treatment of clostridial ferredoxin with cyanide caused bleaching of the protein and formation of thiocyanate. The rate of bleaching was increased by urea, heat, or alkali. In experiments with C. acidi-urici [³⁵S]sulfide-ferredoxin, it was shown that cyanolysis converts 70–80% of the sulfide to [³⁵S]thiocyanate. Apoferredoxin_ox, other disulfides, or Na₂S alone did not yield thiocyanate under these conditions. However, the apoprotein, as well as 2-mercaptoethanol disulfide, forms thiocyanate when Na₂S is added. The addition of Na₂S also increases the amount of thiocyanate formed from ferredoxin. The specific activity of the thiocyanate formed from [³⁵S]sulfide-ferredoxin in the presence of added unlabeled Na₂S is greatly decreased. The specific activity of the thiocyanate formed from the cyanolysis of [³⁵S]sulfide-ferredoxin in the presence of urea and excess sulfide increased with time. Bleaching of ferredoxin during cyanolysis in the presence of urea led to the release of inorganic sulfide prior to the formation of thiocyanate. These observations suggest that it is likely that thiocyanate formation from ferredoxin and cyanide results from the production of a persulfide bond between the apoprotein and the released sulfide. Therefore, thiocyanate production from ferredoxin treated with cyanide does not constitute evidence for the occurrence of the persulfide group in the native protein.     

Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and acetate, both excreted fermentation products of the bivalve, was tested.Anoxic incubations with seawater diluted with demineralised water showed at the lowest salinity (50% seawater, SW) a significant increase in the capacity of M. edulis to survive anoxia as compared to both 75% SW and control [100% SW, corresponding to 32 practical salinity units (psu)]. Formation of biotic sulphide and ammonium occurred in all incubations. However, bacterial proliferation was postponed by 2-3 days at lowest salinity and accordingly, concentrations of both compounds were lower. Anoxic survival profiles of mussels collected from different habitats in the Dutch Scheldt area, characterised by differences in salinity (range from 17 to 31 psu), corresponded with the above salinity effect. Walsoorden mussels (17 psu) showed the longest (P<0.001) survival time under anoxia (LT₅₀=17.2 days) as compared with Paulina (27 psu) and Wemeldinge (31 psu) mussels (LT₅₀=12.8 and 9.8 days, respectively). Condition index (ratio of soft body weight to shell volume) was not correlated with anoxic survival time in untreated mussels, although this was clearly the case when the antibiotic chloramphenicol was added to the anoxic seawater.Acidification of the anoxic incubation medium had a positive effect on survival time. LT₅₀ values significantly (P<0.001) increased from 10.2 days at pH 8.1 to 11.6 and 11.5 days at pH 7.3 and 6.5, respectively. Biotic sulphide and ammonium accumulation as well as bacterial numbers were significantly lower at pH 7.3 and 6.5 as compared with pH 8.1. Anoxic incubations at 10 °C (LT₅₀=12.0 days) strongly increased survival time as compared to 18 °C (LT₅₀=5.9 days). The benefit of antibiotic addition was also stronger at lower temperature (10 °C).Addition of both propionate and acetate (0.5 mM) displayed no effect on mortality of mussels under anoxia, but ammonium (0.5 mM) caused a negative effect (P<0.001). Biotic sulphide and ammonium concentrations measured in both volatile fatty acid incubations were lower than the control situation, as well as total bacterial numbers.This study shows that environmental factors play a significant role in determining the course of bacterial infection and death of bivalves exposed to anoxia.     

Biochemical properties of nematode O-acetylserine(thiol)lyase paralogs imply their distinct roles in hydrogen sulfide homeostasis

O-Acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in a sulfur assimilation pathway incorporating sulfide into amino acids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1–cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get an insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.     

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.     

An attempt to restore suitable conditions for demersal fishes and crustaceans in the Port of Sakai-Semboku, north Osaka Bay, Japan

Hypoxia and/or anoxia developed in the summer of 1995 and 1996 in the lower water layer in the Port of Sakai-Semboku, north Osaka Bay, causing an increase in sulfide content and a decrease in the redox potential of the bottom sediment. As a result, the number of benthic animal species was reduced and in the innermost part of the port no benthic animals were found. A jet stream pump system was installed at the Dejima Fishing Port, the innermost part of the Port of Sakai-Semboku to improve the oxygen conditions in the bottom waters. This led to an increase in percentage saturation of oxygen (ca. 10% increase) of the lower seawater layer along with an increase in richness and abundance of benthic fishes and crustaceans in the summer. These findings indicate that dissolved oxygen is a crucial environmental factor in the functioning of the port as a habitat for various benthic animals in north Osaka Bay. Based on the results of field surveys, the possibility of restoration of the benthic fauna by using a jet stream pump system is also discussed.     

Understanding the Anodic Mechanism of a Seafloor Fuel Cell: Interactions Between Geochemistry and Microbial Activity

Seafloor fuel cells made with graphite electrodes generate electricity by promoting electron transfer in response to a natural voltage difference (−0.7 to −0.8 V) between anoxic sediments and overlying oxic seawater. Geochemical impacts of a seafloor fuel cell on sediment solids and porewaters were examined to identify the anodic mechanisms and substrates available for current production. In an estuarine environment with little dissolved sulfide, solid-phase acid volatile sulfide and Cr²⁺-reducible sulfur minerals decreased significantly toward the anode after 7 months of nearly continuous energy harvesting. Porewater iron and sulfate increased by millimolar amounts. Scanning electron microscope images showed a biofilm overcoating the anode, and electron microprobe analyses revealed accumulations of sulfur, iron, silicon and phosphorus at the electrode surface. Sulfur deposition was also observed on a laboratory fuel cell anode used to generate electricity with only dissolved sulfide as an electron donor. Moreover, current densities and voltages displayed by these purely chemical cells were similar to the values measured with field devices. These results indicate that electron transfer to seafloor fuel cells can readily result in the oxidation of dissolved and solid-phase forms of reduced sulfur producing mainly S⁰ which deposits at the electrode surface. This oxidation product is consistent with the observed enrichment of bacteria most closely related to Desulfobulbus/Desulfocapsa genera within the anode biofilm, and its presence is proposed to promote a localized biogeochemical cycle whereby biofilm bacteria regenerate sulfate and sulfide. This electron-shuttling mechanism may co-occur while these or other bacteria use the anode directly as a terminal electron acceptor.     

In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K_m, 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V_max for DMSP lyase was 34.7 mU · mg of protein⁻¹. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.     

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.     

RESPONSES OF GYMNODINIUM BREVE DAVIS TO NATURAL WATERS OF DIVERSE ORIGIN1

The effect on the growth of Gymnosdinium breve of river waters and seawater samples collected in different seasons and locations was investigated with and without differential enrichments. Growth in natural seawater was most clearly enhanced by addition of EDTA-Fe, sulfide, N and P. The seasonal variation in growth-promoting properties of seawater and river water dominates variations due to differences in location.     

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.     

Light Actions in the Germination of Cocklebur Seeds: IV. DISAPPEARANCE OF RED LIGHT-REQUIREMENT FOR THE GERMINATION OF UPPER SEEDS SUBJECT TO ANOXIA, CHILLING, CYANIDE OR AZIDEPRETREATMENT

Esashi, Y., Fuwa, Nn Kojima, K. and Hase, S. 1986. Light actionsin the germination of cocklebur seeds. IV. Disappearance ofred light-requirement for the germination of upper seeds subjectto anoxia, chilling, cyanide or azide pretreatmenL—J.exp. Bot. 37: 1652–1662. The effects on the germination of positively photoblastic uppercocklebur (X anthium pennsylvanicum Wallr.) seeds by pretreatingwith anoxia, chilling, cyanide or azide, which stimulates theirdark germination, were examined in relation to light actions.Prior to experiments, seeds were pre-soaked at 23 °C inthe dark for 1 or 2 weeks to remove the pre-existing Pfr. Whenthe prctreatment conditions were suboptimal for germinationinduction, the stimulating effects of the pretreatments on germinationduring a subsequent dark period at 23 °C were manifest onlywhen seeds were irradiated with red light before or after thepretreatment Red light promotion was reversed by blue or far-redlight treatment. However, both prc-chilling for 6 d at 8 °Cand prctreatment with 1· 5 mol m ^{– 3} NaN³ for 2d could induce full germination without red light exposure.On the other hand, both pre-exposure to anoxia for 8 d and pretreatmentwith 30 mol m^–3 KCN could induce the dark germinationonly when germination occurred at 33 °C which is known toaugment the ratio of an alternative respiration flux to a cytochromeone. Moreover, the dark germination in response to these inductionswere strongly inhibited by the inhibitors of alternative respiration,propyl gallate and benzohydroxamic acid, applied during a subsequentdark period. It was thus suggested that Pfr has some relationto the operation of two respiration systems of cocklebur seeds,but it is not indispensable to germination of this positivelyphotoblastic seed. Key words: Anoxia, azide, blue light, chilling cyanide, dark germination, far-red light, red light, seed germination, X anthium pennsylvanicum     

Abundance and Distribution of Dimethylsulfoniopropionate Degradation Genes and the Corresponding Bacterial Community Structure at Dimethyl Sulfide Hot Spots in the Tropical and Subtropical Pacific Ocean

Dimethylsulfoniopropionate (DMSP) is mainly produced by marine phytoplankton but is released into the microbial food web and degraded by marine bacteria to dimethyl sulfide (DMS) and other products. To reveal the abundance and distribution of bacterial DMSP degradation genes and the corresponding bacterial communities in relation to DMS and DMSP concentrations in seawater, we collected surface seawater samples from DMS hot spot sites during a cruise across the Pacific Ocean. We analyzed the genes encoding DMSP lyase (dddP) and DMSP demethylase (dmdA), which are responsible for the transformation of DMSP to DMS and DMSP assimilation, respectively. The averaged abundance (±standard deviation) of these DMSP degradation genes relative to that of the 16S rRNA genes was 33% ± 12%. The abundances of these genes showed large spatial variations. dddP genes showed more variation in abundances than dmdA genes. Multidimensional analysis based on the abundances of DMSP degradation genes and environmental factors revealed that the distribution pattern of these genes was influenced by chlorophyll a concentrations and temperatures. dddP genes, dmdA subclade C/2 genes, and dmdA subclade D genes exhibited significant correlations with the marine Roseobacter clade, SAR11 subgroup Ib, and SAR11 subgroup Ia, respectively. SAR11 subgroups Ia and Ib, which possessed dmdA genes, were suggested to be the main potential DMSP consumers. The Roseobacter clade members possessing dddP genes in oligotrophic subtropical regions were possible DMS producers. These results suggest that DMSP degradation genes are abundant and widely distributed in the surface seawater and that the marine bacteria possessing these genes influence the degradation of DMSP and regulate the emissions of DMS in subtropical gyres of the Pacific Ocean.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

Cardiovascular anatomy and cardiac function in the air-breathing swamp eel (Monopterus albus)

Monopterus albus, a swamp eel inhabiting the freshwaters of South East Asia, relies on an extensive vascularisation of the buccal cavity, pharynx and anterior oesophagus for gas exchange, while the gills are much reduced. In the present study we describe the macro-circulation in the cephalic region and the vascularisation of the buccal cavity of M. albus using vascular fillings and micro-computed tomography (μCT). We also show that M. albus has the capacity to use the buccal cavity for aquatic gas exchange, being able to maintain normal arterial blood gas composition, blood pressure, heart rate and cardiac output throughout 10 h of forced submergence. M. albus therefore can be characterised as a facultative air-breather. Because M. albus aestivates for many months in moist mud during the dry season we characterised in vivo cardiovascular function during exposure to anoxia as well as the effects of anoxia on in vitro contractility of strip preparations from atria and ventricle. Both studies revealed a low anoxia tolerance, rendering it unlikely that M. albus can survive prolonged exposure to anoxia.     

Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O₂ was displaced by continuous infusion of argon. Perfusion with medium at PO₂ < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure. Special issue dedicated to John P. Blass.