Artifical light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species.

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

Comparative genomic analysis identifies small open reading frames (sORFs) with peptide-encoding features in avian 16S rDNA.

The mitochondrial genome (mt-DNA) functional repertoire has recently been enriched in mammals by the identification of functional small open reading frames (sORFs) embedded in ribosomal DNAs. Through comparative genomic analyses the presence of putatively functional sORFs was investigated in birds. Alignment of available avian mt-DNA sequences revealed highly conserved regions containing four putative sORFs that presented low insertion/deletion polymorphism rate (<0.1%) and preserved in frame start/stop codons in >80% of species. Detected sORFs included avian homologs of human Humanin and Short-Humanin-Like-Peptide 6 and two new sORFs not yet described in mammals. The amino-acid sequences of the four putative encoded peptides were strongly conserved among birds, with amino-acid p-distances (5.6 to 25.4%) similar to those calculated for typical avian mt-DNA-encoded proteins (14.8%). Conservation resulted from either drastic conservation of the nucleotide sequence or negative selection pressure. These data extend to birds the possibility that mitochondrial rDNA may encode small bioactive peptides.

Skeletal muscle metabolism in sea-acclimatized king penguins. I. Thermogenic mechanisms.

At fledging, king penguin juveniles undergo a major energetic challenge to overcome the intense and prolonged energy demands for thermoregulation and locomotion imposed by life in cold seas. Among other responses, sea acclimatization triggers fuel selection in skeletal muscle metabolism towards lipid oxidation in vitro, which is reflected by a drastic increase in lipid-induced thermogenesis in vivo However, the exact nature of skeletal muscle thermogenic mechanisms (shivering and/or non-shivering thermogenesis) remains undefined. The aim of the present study was to determine in vivo whether the capacity for non-shivering thermogenesis was enhanced by sea acclimatization. We measured body temperature, metabolic rate, heart rate and shivering activity in fully immersed king penguins (Aptenodytes patagonicus) exposed to water temperatures ranging from 12 to 29°C. Results from terrestrial pre-fledging juveniles were compared with those from sea-acclimatized immature penguins (hereafter 'immatures'). The capacity for thermogenesis in water was as effective in juveniles as in immatures, while the capacity for non-shivering thermogenesis was not reinforced by sea acclimatization. This result suggests that king penguins mainly rely on skeletal muscle contraction (shivering or locomotor activity) to maintain endothermy at sea. Sea-acclimatized immature penguins also exhibited higher shivering efficiency and oxygen pulse (amount of oxygen consumed or energy expended per heartbeat) than pre-fledging juvenile birds. Such increase in shivering and cardiovascular efficiency may favor a more efficient activity-thermoregulatory heat substitution providing penguins with the aptitude to survive the tremendous energetic challenge imposed by marine life in cold circumpolar oceans.

Skeletal muscle metabolism in sea-acclimatized king penguins. II. Improved efficiency of mitochondrial bioenergetics.

At fledging, juvenile king penguins (Aptenodytes patagonicus) must overcome the tremendous energetic constraints imposed by their marine habitat, including during sustained extensive swimming activity and deep dives in cold seawater. Both endurance swimming and skeletal muscle thermogenesis require high mitochondrial respiratory capacity while the submerged part of dive cycles repeatedly and greatly reduces oxygen availability, imposing a need for solutions to conserve oxygen. The aim of the present study was to determine in vitro whether skeletal muscle mitochondria become more 'thermogenic' to sustain heat production or more 'economical' to conserve oxygen in sea-acclimatized immature penguins (hereafter 'immatures') compared with terrestrial juveniles. Rates of mitochondrial oxidative phosphorylation were measured in permeabilized fibers and mitochondria from the pectoralis muscle. Mitochondrial ATP synthesis and coupling efficiency were measured in isolated muscle mitochondria. The mitochondrial activities of respiratory chain complexes and citrate synthase were also assessed. The results showed that respiration, ATP synthesis and respiratory chain complex activities in pectoralis muscles were increased by sea acclimatization. Furthermore, muscle mitochondria were on average 30-45% more energy efficient in sea-acclimatized immatures than in pre-fledging juveniles, depending on the respiratory substrate used (pyruvate, palmitoylcarnitine). Hence sea acclimatization favors the development of economical management of oxygen, decreasing the oxygen needed to produce a given amount of ATP. This mitochondrial phenotype may improve dive performance during the early marine life of king penguins, by extending their aerobic dive limit.

Improved mitochondrial coupling as a response to high mass-specific metabolic rate in extremely small mammals.

Mass-specific metabolic rate negatively co-varies with body mass from the whole-animal to the mitochondrial levels. Mitochondria are the mainly consumers of oxygen inspired by mammals to generate ATP or compensate for energetic losses dissipated as the form of heat (proton leak) during oxidative phosphorylation. Consequently, ATP synthesis and proton leak compete for the same electrochemical gradient. Because proton leak co-varies negatively with body mass, it is unknown whether extremely small mammals further decouple their mitochondria to maintain their body temperature or whether they implement metabolic innovations to ensure cellular homeostasis. The present study investigated the impact of body mass variation on cellular and mitochondrial functioning in small mammals, comparing two extremely small African pygmy mice (Mus mattheyi, ∼5 g, and Mus minutoides, ∼7 g) with the larger house mouse (Mus musculus, ∼22 g). Oxygen consumption rates were measured from the animal to the mitochondrial levels. We also measured mitochondrial ATP synthesis in order to appreciate the mitochondrial efficiency (ATP/O). At the whole-animal scale, mass- and surface-specific metabolic rates co-varied negatively with body mass, whereas this was not necessarily the case at the cellular and mitochondrial levels. Mus mattheyi had generally the lowest cellular and mitochondrial fluxes, depending on the tissue considered (liver or skeletal muscle), as well as having more-efficient muscle mitochondria than the other two species. Mus mattheyi presents metabolic innovations to ensure its homeostasis, by generating more ATP per oxygen consumed.

Threshold effect in the H2O2 production of skeletal muscle mitochondria during fasting and refeeding.

Under nutritional deprivation, the energetic benefits of reducing mitochondrial metabolism are often associated with enhanced harmful pro-oxidant effects and a subsequent long-term negative impact on cellular integrity. However, the flexibility of mitochondrial functioning under stress has rarely been explored during the transition from basal non-phosphorylating to maximal phosphorylating oxygen consumption. Here, we experimentally tested whether ducklings (Cairina moschata), fasted for 6 days and subsequently refed for 3 days, exhibited modifications to their mitochondrial fluxes, i.e. oxygen consumption, ATP synthesis, reactive oxygen species generation (ROS) and associated ratios, such as the electron leak (% ROS/O) and the oxidative cost of ATP production (% ROS/ATP). This was carried out at different steady-state rates of oxidative phosphorylation in both pectoralis (glycolytic) and gastrocnemius (oxidative) muscles. Fasting induced a decrease in the rates of oxidative phosphorylation and maximal ROS release. These changes were completely reversed by 3 days of refeeding. Yet, the fundamental finding of the present study was the existence of a clear threshold in ROS release and associated ratios, which remained low until a low level of mitochondrial activity was reached (30-40% of maximal oxidative phosphorylation activity).

Uncoupling effect of palmitate is exacerbated in skeletal muscle mitochondria of sea-acclimatized king penguins (Aptenodytes patagonicus).

In king penguin juveniles, the environmental transition from a terrestrial to a marine habitat, occurring at fledging, drastically stimulates lipid catabolism and the remodelling of muscle mitochondria to sustain extensive swimming activity and thermoregulation in the cold circumpolar oceans. However, the exact nature of these mechanisms remains only partially resolved. Here we investigated, in vitro, the uncoupling effect of increasing doses of fatty acids in pectoralis muscle intermyofibrillar mitochondria isolated, either from terrestrial never-immersed or experimentally cold water immersed pre-fledging king penguins or from sea-acclimatized fledged penguins. Mitochondria exhibited much greater palmitate-induced uncoupling respiration and higher maximal oxidative capacity after acclimatization to marine life. Such effects were not reproduced experimentally after repeated immersions in cold water, suggesting that the plasticity of mitochondrial characteristics may not be primarily driven by cold exposure per se but by other aspects of sea acclimatization.

Underestimated contribution of skeletal muscle in ornithine metabolism during mouse postnatal development.

Ornithine aminotransferase (L-ornithine 2-oxoacid aminotransferase, OAT) is widely expressed in organs, but studies in mice have focused primarily on the intestine, kidney and liver because of the high OAT-specific activity in these tissues. This study aimed to investigate OAT activity in adult mouse tissues to assess the potential contribution to ornithine metabolism and to determine OAT control during postnatal development. OAT activity was widely distributed in mouse tissues. Sexual dimorphism was observed for most tissues in adults, with greater activity in females than in males. The contribution of skeletal muscles to total OAT activity (34% in males and 27% in females) was the greatest (50%) of the investigated tissues in pre-weaned mice and was similar to that of the liver in adults. OAT activity was found to be regulated in a tissue-specific manner during postnatal development in parallel with large changes in the plasma testosterone and corticosterone levels. After weaning, OAT activity markedly increased in the liver but dropped in the skeletal muscle and adipose tissue. Anticipating weaning for 3 days led to an earlier reduction of OAT activity in skeletal muscles. Orchidectomy in adults decreased OAT activity in the liver but increased it in skeletal muscle and adipose tissue. We concluded that the contribution of skeletal muscle to mouse ornithine metabolism may have been underestimated. The regulation of OAT in skeletal muscles differs from that in the liver. The present findings suggest important and tissue-specific metabolic roles for OAT during postnatal development in mice.

Ontogeny of muscle bioenergetics in Adelie penguin chicks (Pygoscelis adeliae).

The ontogeny of pectoralis muscle bioenergetics was studied in growing Adélie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvate-malate-succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxyl-acyl-CoA dehydrogenase) increased 6- to 19-fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3- to 5-fold) and oxidative activities (1.5- to 2.4-fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates, suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), were detected by Western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.

Selective upregulation of lipid metabolism in skeletal muscle of foraging juvenile king penguins: an integrative study.

The passage from shore to marine life of juvenile penguins represents a major energetic challenge to fuel intense and prolonged demands for thermoregulation and locomotion. Some functional changes developed at this crucial step were investigated by comparing pre-fledging king penguins with sea-acclimatized (SA) juveniles (Aptenodytes patagonicus). Transcriptomic analysis of pectoralis muscle biopsies revealed that most genes encoding proteins involved in lipid transport or catabolism were upregulated, while genes involved in carbohydrate metabolism were mostly downregulated in SA birds. Determination of muscle enzymatic activities showed no changes in enzymes involved in the glycolytic pathway, but increased 3-hydroxyacyl-CoA dehydrogenase, an enzyme of the ß-oxidation pathway. The respiratory rates of isolated muscle mitochondria were much higher with a substrate arising from lipid metabolism (palmitoyl-L-carnitine) in SA juveniles than in terrestrial controls, while no difference emerged with a substrate arising from carbohydrate metabolism (pyruvate). In vivo, perfusion of a lipid emulsion induced a fourfold larger thermogenic effect in SA than in control juveniles. The present integrative study shows that fuel selection towards lipid oxidation characterizes penguin acclimatization to marine life. Such acclimatization may involve thyroid hormones through their nuclear beta receptor and nuclear coactivators.

Transcriptome-wide deregulation of gene expression by artificial light at night in tadpoles of common toads.

Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by potentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global effect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experiment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tadpoles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to controls, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and during the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmental immune challenges.

Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays.

BACKGROUND: Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. RESULTS: We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. CONCLUSIONS: MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

Cold-acclimation-induced non-shivering thermogenesis in birds is associated with upregulation of avian UCP but not with innate uncoupling or altered ATP efficiency.

Despite their lack of brown adipose tissue, some bird species develop regulatory non-shivering thermogenesis (NST) of skeletal muscle origin in response to cold acclimation. Mechanisms involved in avian NST are still unclear but may involve reduced energetic coupling in skeletal muscle mitochondria through the expression of an avian homologue of mammalian uncoupling proteins. The aim of this work was to investigate whether the expression of avian uncoupling protein (avUCP) would correlate with the capacity for cold-induced muscle NST. Various levels of cold acclimation were obtained by rearing 1-week-old ducklings (Cairina moschata) for 4 weeks at three different ambient temperatures (25 degrees C, 11 degrees C or 4 degrees C). Muscle NST was measured by simultaneous recordings of metabolic rate and electromyographic activity (gastrocnemius muscle) at ambient temperatures (T(a)) ranging from 27 degrees C to -5 degrees C. The expression of avUCP gene and mitochondrial bioenergetics were also determined in gastrocnemius muscle. Results showed that muscle NST capacity depends on the T(a) at which ducklings were acclimated, i.e. the lower the rearing temperature, the higher the capacity for NST. This increased metabolic heat production occurred in parallel with an upregulation of avUCP, which was not associated with a change in mitochondrial membrane conductance. The intensity of mitochondrial oxidative phosphorylation also increased in proportion with the harshness of cold, while the efficiency of ATP generation was equally effective in all three acclimation temperatures. In the absence of mitochondrial uncoupling, these data indicate a clear link between avUCP expression and the capacity of ducklings to adjust their muscular aerobic activity to cold exposure.

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria.

BACKGROUND: Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. RESULTS: The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase). CONCLUSION: This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

Artificial light at night alters the sexual behaviour and fertilisation success of the common toad.

Artificial Light At Night (ALAN) is an emerging pollution, that dramatically keeps on increasing worldwide due to urbanisation and transport infrastructure development. In 2016, it nearly affected 23% of the Earth's surface. To date, all terrestrial and aquatic ecosystems have been affected. The disruption of natural light cycles due to ALAN is particularly expected for nocturnal species, which require dark periods to forage, move, and reproduce. Apart from chiropterans, amphibians contain the largest proportion of nocturnal species among vertebrates exhibiting an unfavourable conservation status in most parts of the world and living in ALAN polluted areas. Despite the growing number of studies on this subject, our knowledge on the direct influence of nocturnal lighting on amphibians is still scarce. To better understand the consequences of ALAN on the breeding component of amphibian fitness, we experimentally exposed male breeding common toads (Bufo bufo) to ecologically relevant light intensities of 0.01 (control), 0.1 or 5 lux for 12 days. At mating, exposed males took longer than controls to form an amplexus, i.e. to pair with a female, and broke amplexus before egg laying, while controls never did. These behavioural changes were associated with fitness alteration. The fertilisation rate of 5 lux-exposed males was reduced by 25%. Salivary testosterone, which is usually correlated with reproductive behaviours, was not altered by ALAN. Our study demonstrates that ALAN can affect the breeding behaviour of anuran species and reduce one component of their fitness. Given the growing importance of ALAN, more work is needed to understand its long-term consequences on the behaviour and physiology of individuals. It appears essential to identify deleterious effects for animal populations and propose appropriate management solutions in an increasingly brighter world.

Stimulating fermentation by the prolonged acceleration of gut transit protects against decompression sickness.

Massive bubble formation after diving can lead to decompression sickness (DCS). Gut fermentation at the time of a dive exacerbates DCS due to endogenous hydrogen production. We sought to investigate whether medium-term stimulation of fermentation as a result of polyethylene glycol (PEG)-induced acceleration of bowel transit before diving exacerbates DCS in rats. Seven days before an experimental dry dive, 60 rats were randomly divided in two groups: an experimental group treated with PEG (n = 30) and an untreated control group (n = 30). Exhaled hydrogen was measured before the dive. Following hyperbaric exposure, we assessed for signs of DCS. After anaesthetisation, arterial blood was drawn to assay inflammatory cytokines and markers of oxidative stress. PEG led to a significant increase in exhaled H2 (35 ppm [10-73] compared with control 7 ppm [2-15]; p = 0.001). The probability of death was reduced in PEG-treated rats (PEG: 17% [95% CI 4-41] vs control: 50% [95% CI 26-74]; p = 0.034). In addition, inflammatory markers were reduced, and the antioxidant activity of glutathione peroxidase was significantly increased (529.2 U.l-1 [485.4-569.0] versus 366.4 U.l-1 [317.6-414.8]; p = 0.004). Thus, gut fermentation might have a positive effect on DCS. The antioxidant and neuroprotective properties of the fermentation by-products H2 and butyrate may explain these results.

Prevention of unloading-induced atrophy by vitamin E supplementation: links between oxidative stress and soleus muscle proteolysis?

Exposure to reduced activity induces skeletal muscle atrophy. Oxidative stress might contribute to muscle wasting via proteolysis activation. This study aimed to test two hypotheses in rats. First, supplementation of the antioxidant vitamin E, prior and during the phase of unloading, would partly counteract unloading-induced soleus muscle atrophy. Secondly, vitamin E supplementation would decrease the rate of muscle proteolysis by reducing expression of calpains, caspases-3, -9, and -12, and E3 ubiquitin ligases (MuRF1 and MAFbx). Soleus muscle atrophy (-49%) induced by 14 days of hindlimb unloading was reduced to only 32% under vitamin E. Vitamin E partly prevented the decrease in type I and IIa fiber size. Supplementation increased HSP72 content and suppressed the rise in muscle level of thiobarbituric acid-reactive substance caused by unloading but failed to modify the lower ratio of reduced vs oxidized glutathione, the higher uncoupling proteins mRNA, and the antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase) observed after unloading. Vitamin E treatment abolished the large upregulation of caspases-9 and -12 and MuRF1 transcripts in unloaded muscle and greatly decreased the upregulation of mu-calpain, caspase-3, and MAFbx mRNA. In conclusion, the protective effect of vitamin E might be due to modulation of muscle proteolysis-related genes rather than to its antioxidant function.

Transcriptomic data analysis and differential gene expression of antioxidant pathways in king penguin juveniles (Aptenodytes patagonicus) before and after acclimatization to marine life.

In this article, we present differentially expressed gene profiles in the pectoralis muscle of wild juvenile king penguins that were either naturally acclimated to cold marine environment or experimentally immersed in cold water as compared with penguin juveniles that never experienced cold water immersion. Transcriptomic data were obtained by hybridizing penguins total cDNA on Affymetrix GeneChip Chicken Genome arrays and analyzed using maxRS algorithm, "Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays" (Dégletagne et al., 2010) [1]. We focused on genes involved in multiple antioxidant pathways. For better clarity, these differentially expressed genes were clustered into six functional groups according to their role in controlling redox homeostasis. The data are related to a comprehensive research study on the ontogeny of antioxidant functions in king penguins, "Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)" (Rey et al., 2016) [2]. The raw microarray dataset supporting the present analyses has been deposited at the Gene Expression Omnibus (GEO) repository under accessions GEO: GSE17725 and GEO: GSE82344.

Colonic Fermentation Promotes Decompression sickness in Rats.

Massive bubble formation after diving can lead to decompression sickness (DCS). During dives with hydrogen as a diluent for oxygen, decreasing the body's H2 burden by inoculating hydrogen-metabolizing microbes into the gut reduces the risk of DCS. So we set out to investigate if colonic fermentation leading to endogenous hydrogen production promotes DCS in fasting rats. Four hours before an experimental dive, 93 fasting rats were force-fed, half of them with mannitol and the other half with water. Exhaled hydrogen was measured before and after force-feeding. Following the hyperbaric exposure, we looked for signs of DCS. A higher incidence of DCS was found in rats force-fed with mannitol than in those force-fed with water (80%, [95%CI 56, 94] versus 40%, [95%CI 19, 64], p < 0.01). In rats force-fed with mannitol, metronidazole pretreatment reduced the incidence of DCS (33%, [95%CI 15, 57], p = 0.005) at the same time as it inhibited colonic fermentation (14 ± 35 ppm versus 118 ± 90 ppm, p = 0.0001). Pre-diveingestion of mannitol increased the incidence of DCS in fasting rats when colonic fermentation peaked during the decompression phase. More generally, colonic fermentation in rats on a normal diet could promote DCS through endogenous hydrogen production.

Gut fermentation seems to promote decompression sickness in humans.

Massive bubble formation after diving can lead to decompression sickness (DCS) that can result in neurological disorders. In experimental dives using hydrogen as the diluent gas, decreasing the body's H2 burden by inoculating hydrogen-metabolizing microbes into the gut reduces the risk of DCS. In contrast, we have shown that gut bacterial fermentation in rats on a standard diet promotes DCS through endogenous hydrogen production. Therefore, we set out to test these experimental results in humans. Thirty-nine divers admitted into our hyperbaric center with neurological DCS (Affected Divers) were compared with 39 healthy divers (Unaffected Divers). Their last meal time and composition were recorded. Gut fermentation rate was estimated by measuring breath hydrogen 1-4 h after the dive. Breath hydrogen concentrations were significantly higher in Affected Divers (15 ppm [6-23] vs. 7 ppm [3-12]; P = 0.0078). With the use of a threshold value of 16.5 ppm, specificity was 87% [95% confidence interval (CI) 73-95] for association with neurological DCS onset. We observed a strong association between hydrogen values above this threshold and an accident occurrence (odds ratio = 5.3, 95% CI 1.8-15.7, P = 0.0025). However, high fermentation potential foodstuffs consumption was not different between Affected and Unaffected Divers. Gut fermentation rate at dive time seemed to be higher in Affected Divers. Hydrogen generated by fermentation diffuses throughout the body and could increase DCS risk. Prevention could be helped by excluding divers who are showing a high fermentation rate, by eliminating gas produced in gut, or even by modifying intestinal microbiota to reduce fermentation rate during a dive.