Extreme Anoxia Research Articles

Probe into Reverse Operation of Apoptosis Gene

Objective: The programmed death process of cells according to gene coding belongs to apoptotic natural extinction (PCD). The purpose of this study is to explore the phenomenon of “returning to old age and rejuvenating children” in the extreme anoxia, no nutrients and survival in the extreme environment of fish and earthworm. Methods: the adult earthworms were put into the sealed quartz sand or fine yellow sand plastic bottle with humidity of 35-40%70 ml and poured out 100-150 d, then put back into the natural environment (simulated natural plastic basin) and raised 100-150 d, to collect the experimental information. The same object can be observed repeatedly. Results: The earthworms which were closed in the little oxygen-free and nutrition-deficient vials were reduced by autophagy, and the rings and reproductive pores disappeared completely. When they were put back into the natural environment for two or three months, they were all restored to their original morphological structure. Conclusion: Most of the same subjects underwent 1-3 years of cyclic observation. The biological structure was adapted to the changing environment. It was helped by the resonance of many biota and complex stress factors.

MitosRNAs and extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance.

Metabolic adaptations during extreme anoxia in the turtle heart and their implications for ischemia-reperfusion injury

ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5 °C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25 °C) and mouse hearts exposed to 30 min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30 min of ischemia in mouse hearts. High NADH/NAD+ ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals.

Antioxidant capacity and anoxia tolerance in Austrofundulus limnaeus embryos.

Embryos of Austrofundulus limnaeus can tolerate extreme environmental stresses by entering into a state of metabolic and developmental arrest known as diapause. Oxidative stress is ubiquitous in aerobic organisms and the unique biology and ecology of A. limnaeus likely results in frequent and repeated exposures to oxidative stress during development. The antioxidant capacity of A. limnaeus was explored during development by measuring antioxidant capacity due to small molecules and several enzymatic antioxidant systems. Diapause II embryos can survive for several days in 1% hydrogen peroxide without indications of negative effects. Surprisingly, both small and large molecule antioxidant systems have the highest capacity during early development, which may be due to maternal provisioning. Antioxidant capacity is largely invested in small molecules during early development and in enzymatic systems during late development. The switch in antioxidant mechanisms and decline in small molecule antioxidants during development correlates with the loss of extreme anoxia tolerance.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia.Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia.Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

Extreme anoxia tolerance in crucian carp and goldfish through neofunctionalization of duplicated genes creating a new ethanol-producing pyruvate decarboxylase pathway

Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer’s yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.

Small noncoding RNA expression during extreme anoxia tolerance of annual killifish (Austrofundulus limnaeus) embryos.

Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance.

Mitochondria‐derived Small Non‐coding RNAs in Extreme Anoxia Tolerance

Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out of anoxia in species that can tolerate anoxia may lend new insights into the role that mitochondria play in cellular homeostasis in response to fluctuating levels of oxygen. The objective of this study was to examine the potential role of small RNAs in long‐term vertebrate anoxia tolerance. Expression patterns of small RNAs were determined using RNAseq in the most anoxia tolerant vertebrate, embryos of the annual killifish Austrofundulus limnaeus. Anoxia tolerance varies during development in this species in a way that allows for the comparison of developmental stages with extreme anoxia tolerance to stages with a more typical vertebrate anoxia tolerance. Embryos of 4 different developmental stages were exposed to anoxia, followed by aerobic recovery, and small RNA expression was determined with Illumina RNAseq under aerobic conditions, anoxia, and during aerobic recovery. A large proportion of the differentially expressed small RNAs were found to be encoded in the mitochondrial genome. About 12% of the unique small RNAs identified were encoded within the mitochondrial genome, and around 20% of the highly differentially expressed RNAs in response to anoxia were mitochondrial in origin. Of the differentially expressed sequences mapping to the mitochondrial genome, small RNAs derived from mitochondrial tRNAs were particularly enriched, with sequences up‐regulated during anoxia and aerobic recovery from anoxia. These small RNAs of interest localize to anoxia sensitive tissues, such as the brain and heart, providing further evidence for a potential role in supporting anoxia tolerance. These same small RNAs have been detected in cell cultures derived from A. limnaeus embryos, by RNAseq and in situ hybridization. Cell culture expression patterns corroborated whole‐embryo sequence expression patterns. This work highlights a potential new class of small RNAs derived from the mitochondrial genome that may act to mediate and coordinate the physiological response to anoxia in this species. Further understanding the biology of these mitochondrially‐derived small RNAs and their role in anoxia tolerance may provide novel insight into combating cellular damage induced by heart attack and stroke, two lethal consequences of heart disease in humans.Support or Funding InformationNSF IOS 1354549; NSF GRFP DGE 1057604

Hypoxia and Anoxia Tolerance in the Annual Killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus are routinely exposed to oxygen limitation during development and are extremely tolerant of anoxia. Importantly, tolerance of anoxia is not strictly associated with entrance into metabolic dormancy associated with diapause II, but rather any embryo will respond to anoxia by entering into a state of anoxia-induced quiescence. Hypoxia causes a reduction in the rate of development, reduced heart rates, and reduced capacities for metabolic enzyme activity in both aerobic and anaerobic pathways. Embryos of A. limnaeus begin life as oxyconformers, and transition into oxyregulators near the completion of embryonic development. As this transition occurs, extreme anoxia tolerance is lost. The rate of early development is independent of oxygen partial pressure, despite the fact that the embryos are oxyconformers. This suggests a contribution from anaerobic pathways to support early development. However, the specific pathways supporting this metabolism are unknown. The response of A. limnaeus embryos to hypoxia and anoxia is unique compared to other fishes and most other vertebrates, and thus future studies on this species may lend insight into novel mechanisms that support survival during prolonged oxygen limitation.

Extreme ocean anoxia during the Late Cryogenian recorded in reefal carbonates of Southern Australia

The Neoproterozoic was a time of great change in the Earth's surface and marine environments, including extensive climate variability, the widespread oxygenation of the oceans and the accompanying rise of animal life. However, the timing of ocean oxygenation remains uncertain, particularly in regard to Cryogenian seas, which were disrupted by large periods of global glaciation. Interglacial Cryogenian reef complexes in the Northern Adelaide Fold Belt of Australia contain abundant primary marine dolomite cements. These cements have well-preserved textural and growth zonation, indicating they preserve their original marine chemistry and can be used as geochemical proxies for Late Cryogenian paleooceanography. Analysis of marine cements from peritidal nearshore facies, shallow platformal facies and deep framework facies of the reef complexes reveals significant geochemical gradients with paleo-depth. While nearshore cements have low Fe contents and commonly contain iron-oxide inclusions, the shallow and deep cements have very high Fe concentrations. Chalcophile elements (Cu, Cd, Pb, Zn, etc.) are most abundant in the nearshore cements, whereas rare earth elements are found in highest concentrations in the deeper facies cements. Rare earth element profiles are unusual, with shallow and deep facies having convex profiles with negligible Ce/Ce* anomalies and positive Eu/Eu* anomalies.Being constrained by sedimentology, this carbonate geochemistry provides a window into interglacial Cryogenian ocean chemistry and structure. The marine cements reveal pronounced chemical stratification in this Late Cryogenian ocean. A thin veneer of oxic surface waters existed at the ocean surface, in peritidal facies, with increasingly anoxic and Fe-rich seawater at depth. The distribution of strongly chalcophile elements like Cd and Cu across the chemocline suggests that although ferruginous, deeper anoxic waters probably contained some dissolved sulphide. These conditions describe a ferro-sulfidic ocean and encompass some of the most extreme anoxia yet documented during the late Precambrian. A return to Archean-like ocean conditions at this time suggests large-scale disruption of the ocean system during the Neoproterozoic.

El concepto de aclimatación

Según Coon y Birsdell uno de los factores principales en la diferenciación de los grupos raciales modernos ha sido la selección natural operando en términos y estímulos inherentes a los ambientes extremos. En el cuarto período glacial la raza mongólioa fue la última en diferenciarse desarrollando pechos enormes, extremidades cortas y cuerpos globulares que irradian el menor calor posible. Mongolia es una planicie con una altitud media de 1.500 mts. Los veranos son cálidos y los inviernos fríos. Sus migraciones alcanzan el grado 60 de latitud norte. El ambiente climático corresponde a extremo frío, extrema altitud, y extrema anoxia tal como ocurre en los altiplanos del Himalaya y de los Andes cuyos habitantes tienen las mismas características.

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.

BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Circulating nitric oxide metabolites and cardiovascular changes in the turtleTrachemys scriptaduring normoxia, anoxia and reoxygenation

Turtles of the genus Trachemys show a remarkable ability to survive prolonged anoxia. This is achieved by a strong metabolic depression, redistribution of blood flow and high levels of antioxidant defence. To understand whether nitric oxide (NO), a major regulator of vasodilatation and oxygen consumption, may be involved in the adaptive response of Trachemys to anoxia, we measured NO metabolites (nitrite, S-nitroso, Fe-nitrosyl and N-nitroso compounds) in the plasma and red blood cells of venous and arterial blood of Trachemys scripta turtles during normoxia and after anoxia (3 h) and reoxygenation (30 min) at 21°C, while monitoring blood oxygen content and circulatory parameters. Anoxia caused complete blood oxygen depletion, decrease in heart rate and arterial pressure, and increase in venous pressure, which may enhance heart filling and improve cardiac contractility. Nitrite was present at high, micromolar levels in normoxic blood, as in some other anoxia-tolerant species, without significant arterial-venous differences. Normoxic levels of erythrocyte S-nitroso compounds were within the range found for other vertebrates, despite very high measured thiol content. Fe-nitrosyl and N-nitroso compounds were present at high micromolar levels under normoxia and increased further after anoxia and reoxygenation, suggesting NO generation from nitrite catalysed by deoxygenated haemoglobin, which in turtle had a higher nitrite reductase activity than in hypoxia-intolerant species. Taken together, these data indicate constitutively high circulating levels of NO metabolites and significant increases in blood NO after anoxia and reoxygenation that may contribute to the complex physiological response in the extreme anoxia tolerance of Trachemys turtles.

Characterization of the cell morphology differences between pre‐diapause and diapause embryos of the killifish Austrofundulus limnaeus

Austrofundulus limnaeus is a freshwater annual killifish from Venezuela that produces embryos capable of entering three distinct stages of diapause. Diapause II is a state of developmental arrest that can last over 100 days and confers extreme anoxia tolerance. Diapause I and III are inducible while II is obligate. Diapause II occurs in ideal conditions (25oC) whereas incubation at 30oC induces a bypass of diapause II. Diapause II provides an opportunity to study an arrest of development in a vertebrate animal without exposing the organism to compounding stresses. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state. We hypothesize that down regulation of gene expression is central to inducing diapause II. Our aim is to use assays that are principally based upon immunohistochemistry to characterize at the cellular level differences in cell morphology between pre‐DII and DII embryos. We are looking at changes in chromatin modifications, which are known to be involved in the repression or activation of developmental genes, as well as changes in mitotic cell division. Preliminary results show a change in the signal of H3K27 chromatin modifiers as the animal transitions between stages of pre‐DII and DII. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature, long-term anoxia, desiccation and other wide variety of stressful conditions. How the embryos survive these stresses is a very interesting and unsolved subject. To solve this question we analyzed the nucleotide and deduced protein sequence for Hsp26, a molecular chaperone specific to Artemia embryo development. cDNAs of Hsp26 were sequenced from eight Artemia species and deduced Hsp26 amino acid sequences were analyzed. Computer-assisted analysis indicated that the 5′-untranslated region and all the 3 introns contain many putative cis-acting elements for Hsp26 gene expression during development, including heat shock elements (HSEs), Dfd, dl, CF2-II, Hb and AP-1 binding sites. Secondary structure of the Hsp26 3′-untranslated terminator contains the basic structure basis for transcriptional termination. Hsp26 shares sequence similarity with sHSPs (small heat shock protein) from other organisms. The physico-chemical properties of the deduced protein, such as theoretical molecular weight, protein extinction coefficient, isoelectric point and antigenic sites were also obtained. One seven-peptide nuclear localization signals (NLS) “PFRRRMM” was found, which suggested that the Hsp26 protein was hypothesized to be located inside the nucleus. The numbers of phosphorylation sites of serine, threonine and tyrosine and kinase specific phosphorylation sites are also located in Hsp26 protein sequence. These studies will help us achieve a better understanding of Hsp26 and broad implications for sHSPs function in crustacean embryo development.

Sequential stages in the age-dependent gradual formation and accumulation of tubular aggregates in fast twitch muscle fibers: SERCA and calsequestrin involvement

Tubular aggregates (TAs), ordered arrays of elongated sarcoplasmic reticulum (SR) tubules, are present in skeletal muscle from patients with myopathies and are also experimentally induced by extreme anoxia. In wild-type mice TAs develop in a clear age-, sex- (male), and fiber type- (fast twitch) dependence. However, the events preceding the appearance of TAs have not been explored. We investigated the sequential stages leading to the initial appearance and maturation of TAs in EDL from male mice. TAs' formation requires two temporally distinct steps that operate via different mechanisms. Initially (before 1year of age), the SR Ca(2+) binding protein calsequestrin (CASQ) accumulates specifically at the I band level causing swelling of free SR cisternae. In the second stage, the enlarged SR sacs at the I band level extend into multiple, longitudinally oriented tubules with a full complement of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCA) in the membrane and CASQ in the lumen. Tubules gradually acquire a regular cylindrical shape and uniform size apparently in concert with partial crystallization of SERCA. Multiple, small TAs associate to form fewer mature TAs of very large size. Interestingly, in fibers from CASQ1-knockout mice abnormal aggregates of SR tubules have different conformation and never develop into ordered aggregates of straight cylinders, possibly due to lack of CASQ accumulation. We conclude that TAs do not arise abruptly but are the final result of a gradually changing SR architecture and we suggest that the crystalline ATPase within the aggregates may be inactive.

Mitochondrial physiology of diapausing and developing embryos of the annual killifish Austrofundulus limnaeus: implications for extreme anoxia tolerance

Diapausing embryos of the annual killifish Austrofundulus limnaeus have the highest reported anoxia tolerance of any vertebrate and previous studies indicate modified mitochondrial physiology likely supports anoxic metabolism. Functional mitochondria isolated from diapausing and developing embryos of the annual killifish exhibited VO(2), respiratory control ratios (RCR), and P:O ratios consistent with those obtained from other ectothermic vertebrate species. Reduced oxygen consumption associated with dormancy in whole animal respiration rates are correlated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O ratios for developing embryos were similar to those obtained from adult liver, but were diminished in mitochondria from diapausing embryos suggesting decreased oxidative efficiency. Proton leak in adult liver corresponded with that of developing embryos but was elevated in mitochondria isolated from diapausing embryos. In metabolically suppressed diapause II embryos, over 95% of the mitochondrial oxygen consumption is accounted for by proton leak across the inner mitochondrial membrane. Decreased activity of mitochondrial respiratory chain complexes correlates with diminished oxidative capacity of isolated mitochondria, especially during diapause. Respiratory complexes exhibited suppressed activity in mitochondria with the ATP synthase exhibiting the greatest inhibition during diapause II. Mitochondria isolated from diapause II embryos are not poised to produce ATP, but rather to shuttle carbon and electrons through the Kreb's cycle while minimizing the generation of a proton motive force. This particular mitochondrial physiology is likely a mechanism to avoid production of reactive oxygen species during large-scale changes in flux through oxidative phosphorylation pathways associated with metabolic transitions into and out of dormancy and anoxia.

KILLIFISH: CHAMPION ANOXIA SURVIVORS

The annual killifish Austrofundulus limnaeus lives life on the edge. The Venezuelan ponds where it lives rapidly evaporate in the dry season,killing off the adults and exposing their mud-buried embryos to extended periods of drought. Not that drought is the first of the embryo's problems:microbes living in the soil use up all the oxygen. `Not only are they coping with life without water, but also with life without oxygen,' says Jason Podrabsky from Portland State University, who has investigated with his colleagues the root of the embryos' extreme anoxia tolerance(p. 2253).The key to the embryos' survival is that they go into periods of dormancy,or diapause, twice during their development, even if conditions are ideal.`Their environment is very harsh and unpredictable, so diapause occurs as a part of their natural developmental program,' says Podrabsky. Unlike most annual killifish, A. limnaeus embryos skip diapause I at four days of development, but enter diapause II at around 24 days old and diapause III near the end of development. During diapause the metabolism slows down greatly, so could this be the key to surviving anoxia?To assess anoxia tolerance during development, the team collected fertilised embryos from 4 to 32 days old and put them in embryo medium at 25°C before sealing the jars and bubbling nitrogen through them to remove all the oxygen. They then kept a close eye on the developing embryos, opening the jars when they started dying and counting how many of them were still alive. They found that the later embryos were exposed to anoxia, the longer they survived: 4-day-old embryos suffered 50% mortality after 20 days of anoxia, while 32-day-old embryos, in diapause II, suffered 50% mortality after more than 60 days anoxia. This is the longest anoxia survival of any vertebrate at 25°C.The team knew from the brine shrimp, another anoxia tolerant animal that enters diapause, that there is a window of opportunity after diapause where the animal can shut down again even if it has restarted its development. To find out if this window existed in the killifish embryos, they collected embryos just out of diapause II and tested their anoxia tolerance. They found that embryos exposed to anoxia 4 days after the end of diapause were still very tolerant, but lost this tolerance when they were tested 8 days after,showing that post-diapause safety cushion in the killifish is about 4 days.But what about changes in metabolism? The team took embryos of different developmental stages and flash froze them to preserve their metabolites. After mashing them up and extracting their metabolites, which they analysed using chromatrography and mass spectrometry, they found that anoxic embryos produced lactate, succinate and the amino acid alanine, which are end-products of anaerobic metabolism. This suggests that the embryos survive by relying on glycogen stores and reducing their metabolic rate, but that they haven't developed an alternative strategy for dealing with anoxia. Embryos that accumulated lactate at the slowest rate also survived the longest.They also found that anoxic embryos produced lots of the neurotransmitter GABA, which is known to protect neurons in other anoxia tolerant species, such as turtles and the crucian carp. Podrabsky explains that it's possible that GABA is protecting the traditionally anoxia-sensitive brain and heart tissue in diapause II embryos. For now, the killifish embryo can elbow its way onto the roll-call of champion vertebrate anoxia survivors.

Extreme anoxia tolerance in embryos of the annual killifishAustrofundulus limnaeus: insights from a metabolomics analysis

The annual killifish Austrofundulus limnaeus survives in ephemeral pond habitats by producing drought-tolerant diapausing embryos. These embryos probably experience oxygen deprivation as part of their normal developmental environment. We assessed the anoxia tolerance of A. limnaeus embryos across the duration of embryonic development. Embryos develop a substantial tolerance to anoxia during early development, which peaks during diapause II. This extreme tolerance of anoxia is retained during the first 4 days of post-diapause II development and is then lost. Metabolism during anoxia appears to be supported mainly by production of lactate, with alanine and succinate production contributing to a lesser degree. Anoxic embryos also accumulate large quantities of gamma-aminobutyrate (GABA), a potential protector of neural function. It appears that the suite of characters associated with normal development and entry into diapause II in this species prepares the embryos for long-term survival in anoxia even while the embryos are exposed to aerobic conditions. This is the first report of such extreme anoxia tolerance in a vertebrate embryo, and introduces a new model for the study of anoxia tolerance in vertebrates.

Trace element chemostratigraphy of two Ediacaran–Cambrian successions in South China: Implications for organosedimentary metal enrichment and silicification in the Early Cambrian

A trace element study is presented of two Ediacaran–Early Cambrian sedimentary successions in South China: the Shatan section, Sichuan Province, which represents a shallow platform setting, and the Songtao section, Guizhou Province, which corresponds to a more basinal, depositional environment. Across South China, sedimentary strata of this age exhibit characteristic trace metal enrichments in generally organic-rich, stratigraphically correlative, transgressive units. Trace element (including REE) data are used here to determine the palaeoenvironmental and diagenetic conditions during this Early Cambrian interval of metal scavenging that resulted in enrichments over grey shale of Mo > Cd > V > U >Ni > Ag > Zn > Cu > Pb. At Shatan, high organic carbon contents (up to 5% TOC) correlate with Mo enrichment, which indicates that metal scavenging was linked to organic productivity and decay. Low Th/U ratios are consistent with highly reducing conditions during metal scavenging, while extremely high V contents imply that bottom waters were possibly euxinic at times of extreme anoxia. At Songtao, very low Th/U ratios are strongly associated with high V/Sc and low δ 13C org, implying that anoxia, organic decomposition and metal sequestration were associated in the deepwater facies, too. There is no unambiguous trace element evidence that hydrothermal activity contributed to metal enrichment in South China during the Early Cambrian. The systematic relationship with ocean transgression suggests that enrichment was caused largely by oceanographic changes to organic productivity and burial rather than post-depositional fluid flow. Negative Ce anomalies and seawater-like REE distribution patterns are a feature of the Liuchapo chert and lower black shales of the Jiumenchong Formation at Songtao. Ce depletion indicates that authigenic components of these sedimentary units formed under oxic conditions, which contradicts the other geochemical redox proxies. We argue that this chert derived primarily from biogenic silica from siliceous plankton, which formed above a redox boundary within the water column. Neither silicification nor hydrothermal fluids, contrary to some published models, played a significant role in metal transport.