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Fish red blood cells contain nucleus and therefore carry on active transcription throughout their life. As the steady state level of mRNA depends both on the production and breakdown of the mRNA, it is important to pay attention to sampling, cell separation, mRNA extraction, and transcript stability to conduct a comprehensive erythrocyte genomics study. In addition, particular attention should be made to tie the transcriptional changes to corresponding protein activities, as only those would impact cellular functions. Here, we thus describe the different steps to conduct comprehensive functional genomics studies with fish erythrocytes.

Ectotherms can respond to climate change via evolutionary adaptation, usually resulting in an increase of their upper thermal tolerance. But whether such adaptation influences the phenotypic plasticity of thermal tolerance when encountering further environmental stressors is not clear yet. This is crucial to understand because organisms experience multiple stressors, besides warming climate, in their natural environment and pollution is one of those. Here, we studied the phenotypic plasticity of thermal tolerance in three-spined stickleback populations inhabiting spatially replicated thermally polluted and pristine areas before and after exposing them to a sublethal concentration of copper for one week. We found that the upper thermal tolerance and its phenotypic plasticity after copper exposure did not depend on the thermal history of fish, suggesting that five decades of thermal pollution did not result in evolutionary adaptation to thermal tolerance. The upper thermal tolerance of fish was, on the other hand, increased by ∼ 1.5 °C after 1-week copper exposure in a sex-specific manner, with males having higher plasticity. To our knowledge this is the first study that shows an improvement of the upper thermal tolerance as a result of metal exposure. The results suggest that three-spined sticklebacks are having high plasticity and they are capable of surviving in a multiple-stressor scenario in the wild and that male sticklebacks seem more resilient to fluctuating environmental conditions than female.

The mean value of any parameter and its changes are usually discussed, when ecotoxicological studies are carried out. However, also the variation of any parameter and its changes can be important components of the responses to environmental contamination. Although the homogeneity of variances is commonly tested, testing is done for the use of correct statistical methods, not because of exploring the possibility that variability and its changes could be important components of environmental responses. We evaluated recent aquatic toxicological literature and found that in the majority of articles indicating that homogeneity of variances was tested and giving the result of testing, the assumption of homogeneity was not fulfilled. Further, it was observed that in some studies experimental treatment clearly affected the variability. In this commentary we discuss the reasons for variability: measurement errors, experimental design, genetic heterogeneity and phenotypic plasticity, and conclude that even after accounting for experimental design and genetic makeup significant variability remains. This plasticity may change in environmental responses as suggested by a hypothetical example, and as confirmed by experimental data. As a consequence, the changes of variability can be significant, even when the means do not differ. Because of this, variability and its changes should always be analysed and reported. This will be easy, since the datasets are exactly the same for comparing the variances and means, and as normally variances are tested for homogeneity. It is likely that much new information about the responses of organisms to environmental contamination will be obtained. However, the present journal practises tend to discourage one from concentrating on anything but the mean. In contrast, we think it is imperative that variability is always included as an endpoint in data analysis in the future.

The fitness and recruitment of fish stocks can be markedly affected by environmental disturbances including global warming, eutrophication and contamination. Understanding the effects of environmental stressors on salmon physiology during marine residence is of a global concern as marine survival has decreased. We present a unique combination of physiological responses - antioxidant defence and oxidative damage biomarkers, stable isotopes and contaminant exposure biomarkers - measured from adult Atlantic salmon (Salmo salar) collected at the Baltic Sea and studied in relation to environmental variables and fitness estimates. The results demonstrate that feeding populations of salmon display marked temporal and spatial variation in oxidative status. Better oxidative status of salmon was characterized by a higher amount of reduced glutathione (GSH) and decreased lipid peroxidation (LPX), when the weight-at-age of 3–4-year old sprats was higher and contaminant exposure biomarker (EROD) was lower. Summer season conditions, which included cooler sea surface temperature (SST), higher bottom O2 and less cyanobacteria also indicated conditions for better oxidative status. Summer SST was additionally shown to affected glutathione metabolism enzyme activities. Oxidative status was associated with stable isotopes δ13C and δ15N indicating indirect effect of abiotic conditions and lower levels of the food web. Differences in condition factor and growth were associated with oxidative status in one and two sea winter salmon, respectively. Wild salmon survival was higher in years when they had higher GSH and catalase activity and lower LPX. Enhanced glutathione metabolism and increased protein carbonyls were associated with higher occurrence of yolk-sac fry mortality (M74). Our results show that oxidative status can provide information on exposure to complex combinations of environmental conditions and stressors in the wild and provide a link of physiological function to individual and population level fitness effects. peerReviewed

The interplay of transcriptional and post-transcriptional processes in the regulation of gene expression has been extensively studied in mammals but little is known in other vertebrates so far. Most non-mammalian vertebrates are faced with environmental cues and stressors distinct from those experienced by mammals and thus it is likely that the gene expression strategies differ from those of mammals. Here we performed experiments to study in vitro the various levels of gene expression regulation in nucleated fish red blood cells. Three critical environmental cues frequently experienced by fish were chosen: exposure to hypoxia (2.5%), ambient water temperature increase by 10°C (from 10°C to 20°C), and exposure to stress hormones (represented by the β-adrenergic agonist isoproterenol). We found that β-adrenergic stimulation increases the stability of the β3b-adrenergic receptor (β3b-ar) mRNA, suggesting that mRNA stability can play a role in the regulation of hormonal stress responses in fish. The β3b-ar gene encodes a unique β-adrenergic receptor subtype in fish red blood cells which controls the β-Na+/H+ exchanger activity - an important component of responses to oxygen limitations. Our results furthermore show a yet undescribed link between the Hif1a signaling pathway and the β-adrenergic receptor response. After β-adrenergic stimulation, the transcription of hif1a was activated significantly after 4 hours of exposure. So far, such a response has only been described from mammalian species. This indicates that the β-AR is fundamental to the molecular and physiological responses to hypoxia and that Hif1a might have additional functions than those already known.

Oil spills pose a threat to aquatic organisms. However, the physiological effects of crude oil on cardiac function and on thermal tolerance of juvenile fish are still poorly understood. Consequently, in this paper, we will present results of two separate experiments where we exposed juvenile rainbow trout and European sea bass to crude oil and made cardiac thermal tolerances and maximum heart rate (f Hmax) measurements after 1 week (rainbow trout) and 6-month recovery (sea bass). In both species, the f Hmax was lower in crude oil-exposed fish than in the control ones at temperatures below the optimum but this difference disappeared at higher temperatures. More importantly, the oil-exposed fish had significantly higher Arrhenius break point temperature for f Hmax, which gave an estimate for optimum temperature, than the control fish in both species even though the exposure conditions and recovery times differed between species. The results indicated that exposure of juvenile fish to crude oil did not have a significant negative impact upon their cardiac performance in high temperatures and upper thermal tolerance increased when the fish were tested 1 week or 6 months after the exposure. Our findings suggest that the cardiac function and thermal tolerance of juvenile fish are relatively resistant to a crude oil exposure.

Heinonen, Ilkka, Olli Vuolteenaho, Juha Koskenvuo, Olli Arjamaa, and Mikko Nikinmaa. Systemic hypoxia increases circulating concentration of apelin in humans. High Alt Med Biol. 18:292-295, 2017.Apelin is a hormone that regulates cardiovascular function, and its concentration is increased by hypoxia based on cell culture and animal studies. As it remains unknown as to whether hypoxia could affect apelin levels in humans, we investigated whether breathing normobaric hypoxic gas mixture increases the circulating apelin concentration in healthy male subjects.Ten healthy young men (age 29 ± 5 years, body mass index 24.7 ± 2.8 kg/mArterial oxygen saturation decreased steadily from a baseline value of 99% ± 1% after the initiation hypoxia challenge and reached a steady-state level of 73% ± 6% within 20-30 minutes. Baseline apelin concentration was 3.3 ± 1.3 pmol/L and remained comparable (3.3 ± 1.4 pmol/L) to the baseline concentration at a 2-hour time point. However, apelin concentration at the 24-hour time point (5.5 ± 2.8 pmol/L) was significantly (by ∼67%) higher as compared with at both baseline and 2-hour time points (p 0.05).In conclusion, in line with cell culture and animal studies, acute systemic hypoxia increases circulating apelin concentration also in humans.

Understanding gene expression changes over the lifespan of cells is of fundamental interest and gives important insights into processes related to maturation and aging. This study was undertaken to understand the global transcriptome changes associated with aging in fish erythrocytes. Fish erythrocytes retain their nuclei throughout their lifetime and they are transcriptionally and translationally active. However, they lose important functions during their lifespan in the circulation. We separated rainbow trout (Oncorhynchus mykiss) erythrocytes into young and old fractions using fixed angle-centrifugation and analyzed transcriptome changes using RNA sequencing (RNA-seq) technology and quantitative real-time PCR. We found 930 differentially expressed between young and old erythrocyte fractions; 889 of these showed higher transcript levels in young, while only 34 protein-coding genes had higher transcript levels in old erythrocytes. In particular genes involved in ion binding, signal transduction, membrane transport, and those encoding various enzyme classes are affected in old erythrocytes. The transcripts with higher levels in old erythrocytes were associated with seven different GO terms within biological processes and nine within molecular functions and cellular components, respectively. Our study furthermore found several highly abundant transcripts as well as a number of differentially expressed genes (DEGs) for which the protein products are currently not known revealing the gaps of knowledge in most non-mammalian vertebrates. Our data provide the first insight into changes involved in aging on the transcriptional level and thus opens new perspectives for the study of maturation processes in fish erythrocytes.

The importance of interindividual variability in environmental responses has been little studied, although the available information suggests that, e.g., changes in environmental temperature may be associated with changes in variability. We studied, if exposure to water-soluble fraction (WSF) of crude oil can be associated with changes in interindividual variability in phenotype in Daphnia magna, which reproduces parthenogenetically. By using these clonal organisms, we could exclude the possibility that the observed changes were caused by genetic variability. The results show that the variability of oxygen consumption rate decreased in 48 h 30% WSF-exposed animals as compared to 10% WSF-exposed or control animals without a change in the mean of oxygen consumption rate. The clonal Daphnia magna could also be used to study transgenerational effects without genetic contribution, as the different generations are genetically identical. We observed that the oxygen consumption rates in F1 and F2 generations of unexposed and 10% WSF-exposed Daphnia had decreased from parental F0 generation and were also lower than in offspring of 30% WSF-exposed specimens. The studies did not aim at environmental realism but were designed to show the possibility of variability changes without changes in the mean value of a parameter, and transgenerational effects as a result of environmental contamination.

Aim We studied whether available oxygen without induced mechanical stretch regulates the release of the biologically active B-type natriuretic peptide (BNP) from Langendorff heart. Methods Rat hearts were isolated and perfused with a physiological Krebs–Henseleit solution at a constant hydrostatic pressure in Langendorff set-up. The basal O2 level of perfusate (24.4 ± 0.04 mg L−1) was gradually lowered to 3.0 ± 0.01 mg L−1 over 20 min using N2 gas (n = 7). BNP and O2 level were measured from coronary flow. During control perfusions (n = 5), the O2 concentration was kept at 26.6 ± 0.3 mg L−1. Results A low oxygen concentration in the perfusate was associated with a significant increase in BNP release (F = 40.4, P

Phthalate esters are plasticizers frequently found in wastewater effluents. Previous studies on phthalates have reported anti-androgenic activity in mammals, causing concerns of their potential effects on the reproduction of aquatic organisms. Another group of environmental endocrine disrupters, steroidal estrogens, are known to inhibit steroid biosynthesis in the gonads, but the effects related to spermatogenesis are not well understood in fish. In this study, three-spined sticklebacks were exposed to di-n-butyl phthalate (DBP) and 17α ethinyl-oestradiol (EE2) at nominal concentrations 35μg/L and 40ng/L, respectively, for four days. The aim of the study was to obtain insight into the acute transcriptional responses putatively associated with endocrine disruption. RNA samples from eight individual male fish per treatment (including controls) were used in microarray analysis, covering the expression of approximately 21,000 genes. In the EE2 treatment the results show transcriptional downregulation of genes associated with steroid biosynthesis pathway and up-regulation of genes involved in pathways related to epidermal growth factor signaling and xenobiotic metabolism. The transcriptional response to DBP was in general weaker than to EE2, but based on enrichment analysis, we suggest adverse effects on retinoid metabolism, creatine kinase activity and cell adhesion. Among the genes showing highest fold changes after DBP treatment compared to control was the teleost fish -specific cytochrome P450 17A2. Overall, this study promotes our understanding on molecular responses to anti-androgens and estrogens in fish testes.

Aquatic organisms face multiple stressors in natural ecosystems. More and more often painkillers are detected in surface waters since their prescription has increased worldwide within the last years. Here we examined the effects of the non-steroidal anti-inflammatory drug (NSAID) diclofenac and hypoxia on three-spined sticklebacks (Gasterosteus aculeatus). We exposed sticklebacks to an environmentally relevant concentration of diclofenac (1μg/L) for 14days, to 24h of hypoxia (2.0mg O2/L), and a combination of both. Hypoxia and diclofenac both can be associated with oxidative stress in fish, but it is unclear whether they would act synergistically. Expression analysis of genes related to antioxidant response, hypoxia response, and chemical metabolism in gills showed that diclofenac alone had little effect, while the combination of hypoxia and diclofenac affected transcript levels most, indicating synergistic effects of these stressors. Of the antioxidant enzymes, only superoxide dismutase activity remained unchanged by treatments, while glutathione peroxidase (GPx) was the most affected antioxidant response on both the transcript and activity levels. Our results suggest that diclofenac may lead to suppressed catalase (CAT) activity but increased GPx activity, probably as compensatory mechanism to remove increasing H2O2 in the gills, and that this response is not affected by hypoxia. The activities of lactate dehydrogenase, CAT, and GPx also showed temporal variability during treatments, which can be attributable to tissue-specific circadian rhythms. Our study shows how responses to NSAIDs and hypoxia can interact in fish, suggesting that getting more insight into temporal variation and about the different levels of regulation of environmental responses is necessary in future studies.

The blood of fish has a continuous age distribution of erythrocytes. The properties of young and old erythrocytes differ with young erythrocytes being functionally much more versatile than old erythrocytes, which have higher haemoglobin content. Factors which affect the formation and breakdown of erythrocytes are reviewed. Erythropoiesis in fish is largely similar to that in mammals. However, definitive erythrocytes are mainly formed in the anterior part of the kidney, and erythropoietin is secreted mainly from the heart. Senescence of erythrocytes in fish has not been studied in detail, and consecutively the factors causing aging of erythrocytes in man are discussed. A major factor causing aging of erythrocytes is oxidative stress, which is also a major effect of toxicants on fish erythrocytes. Consequently, apparent age distribution of circulating erythrocytes will be affected by environmental pollution. At present, it is completely unknown, if the changes in age distribution of erythrocytes play a role in pollutant responses or if the age distribution of erythrocytes is actively regulated during environmental adaptation.

Virtually all organisms respond to heat shock by transcription of genes encoding for heat shock proteins (HSPs), but the mechanisms behind post-transcriptional regulation are not known in detail. When we exposed zebrafish to 5 and 7 °C above normal rearing temperature for 30 min, hsp70 mRNA expression was 28 and 150 -fold higher than in control, respectively. Protein expression, on the other hand, showed no significant change at the +5 °C and a 2-fold increase at the +7 °C exposure. This suggests that the transcription of hsp70 gene does not immediately correspond to translation to related proteins under certain stress temperatures, but, when the temperature is higher, and potentially detrimental, transcription and translation are intimately coupled. Those results confirm that temperature is an important abiotic factor involved in heat shock post-transcriptional regulation mechanisms in fish. However, further studies are needed to determine the relationship between this environmental factor and post-transcriptional regulation mechanisms. Earlier, the coupling/uncoupling of hsp transcription and translation has only been studied using cold-water fish, or zebrafish embryos. With current findings, we suggest this mechanism might be present even in adult warm water fish like the zebrafish.

Biological rhythms control the life of virtually all organisms, impacting numerous aspects ranging from subcellular processes to behaviour. Many studies have shown that changes in abiotic environmental conditions can disturb or entrain circadian (∼24 h) rhythms. These expected changes are so large that they could impose risks to the long-term viability of populations. Climate change is a major global stressor affecting the fitness of animals, partially because it challenges the adaptive associations between endogenous clocks and temperature – consequently, one can posit that a large-scale natural experiment on the plasticity of rhythm–temperature interactions is underway. Further risks are posed by chemical pollution and the depletion of oxygen levels in aquatic environments. Here, we focused our attention on fish, which are at heightened risk of being affected by human influence and are adapted to diverse environments showing predictable changes in light conditions, oxygen saturation and temperature. The examined literature to date suggests an abundance of mechanisms that can lead to interactions between responses to hypoxia, pollutants or pathogens and regulation of endogenous rhythms, but also reveals gaps in our understanding of the plasticity of endogenous rhythms in fish and in how these interactions may be disturbed by human influence and affect natural populations. Here, we summarize research on the molecular mechanisms behind environment–clock interactions as they relate to oxygen variability, temperature and responses to pollutants, and propose ways to address these interactions more conclusively in future studies.

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.

Anthropogenic activities are greatly altering the habitats of animals, whereby fish are already encountering several stressors simultaneously. The purpose of the current study was to investigate the capacity of fish to respond to two different environmental stressors (high temperature and overnight hypoxia) separately and together. We found that acclimation to increased temperature (from 7.7±0.02°C to 14.9±0.05°C) and overnight hypoxia (daily changes from normoxia to 63–67% oxygen saturation), simulating climate change and eutrophication, had both antagonistic and synergistic effects on the capacity of fish to tolerate these stressors. The thermal tolerance of Arctic char ( Salvelinus alpinus ) and landlocked salmon ( Salmo salar m. s ebago ) increased with warm acclimation by 1.3 and 2.2°C, respectively, but decreased when warm temperature was combined with overnight hypoxia (by 0.2 and 0.4°C, respectively). In contrast, the combination of the stressors more than doubled hypoxia tolerance in salmon and also increased hypoxia tolerance in char by 22%. Salmon had 1.2°C higher thermal tolerance than char, but char tolerated much lower oxygen levels than salmon at a given temperature. The changes in hypoxia tolerance were connected to the responses of the oxygen supply and delivery system. The relative ventricle mass was higher in cold- than in warm-acclimated salmon but the thickness of the compact layer of the ventricle increased with the combination of warm and hypoxia acclimation in both species. Char had also significantly larger hearts and thicker compact layers than salmon. The results illustrate that while fish can have protective responses when encountering a single environmental stressor, the combination of stressors can have unexpected species-specific effects that will influence their survival capacity.

International audience; Pollution with low concentrations of pharmaceuticals, especially when combined with low-oxygen conditions (hypoxia), is a threat to aquatic ecosystems worldwide. The non-steroidal anti-inflammatory drug diclofenac is commonly detected in wastewater effluents, and has potential to accumulate in the bile of fish. Diclofenac has been shown to activate aryl hydrocarbon receptor (AHR), which induces transcription in the metabolic enzyme cytochrome P450 1a (cyp1a). Previously, crosstalk has been shown to occur between AHR and hypoxia inducible factor 1 (HIF-1). In addition, both of these transcription factors interact with the proteins regulating circadian (24-h) rhythms in vertebrates. Yet little is known about the significance of these interactions during simultaneous exposure to chemicals and hypoxia in fish in vivo. We exposed wild-caught three-spined sticklebacks (Gasterosteus aculeatus) to diclofenac (1 μg/L, 14 days), hypoxia (2.0 mg/L, up to 24 h) and the combination of both. We then analyzed markers of chemical biotransformation (EROD activity, cyp1a and ahr mRNA levels), glycolysis (lactate dehydrogenase (LDH) enzyme activity, ldh and enolase 1a mRNA levels), and the transcription of core circadian clock genes clock and period 1 in liver tissue. Samples were taken at three time points during the light period in order to address disturbances in the circadian variation of metabolic processes. The results show that mRNA levels and LDH activity tended to be lowest before the dark period, but this pattern was disturbed by hypoxia and diclofenac. Diclofenac and hypoxia co-exposure induced EROD activity more strongly than diclofenac exposure alone, while cyp1a mRNA level was increased also by hypoxia and diclofenac alone. LDH activity and mRNA expression showed a clear time-dependent response during hypoxia, which is consistent with the previously suggested decreased accumulation of HIF-1 during the dark period. Furthermore, LDH activity and transcription was disturbed by diclofenac, indicating important effects of environmental pollutants in disturbing natural acclimation. This study demonstrates the need for more studies to understand the potential disturbances in endogenous rhythms caused by environmental pollution in natural populations.

The climate change -driven increase in temperature is occurring rapidly and decreasing the predictability of seasonal rhythms at high latitudes. It is therefore urgent to understand how a change in the relationship between the photoperiod and temperature can affect ectotherms in these environments. We tested whether temperature affects daily rhythms of transcription in a cold-adapted salmonid using high-throughput RNA-sequencing. Arctic char (Salvelinus alpinus) from a subarctic population were reared at a high and a low temperature (15°C and 8°C) for one month under natural, decreasing day length during late summer. Liver transcriptomes were compared between samples collected in the middle and towards the end of the light period and in the middle of the dark period. Daily variation in transcription was lower in fish from the low temperature compared to strong daily variation in warm-acclimated fish, suggesting cold temperatures dampen the cycling of transcriptional rhythms under a simultaneously decreasing day length. Different circadian clock genes had divergent expression patterns, responding either by decreased expression or by increased rhythmicity at 15°C compared to 8°C. The results point out mechanisms that can affect the ability of fish to adapt to increasing temperatures caused by climate change.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

The fitness and reproductive output of fishes can be affected by environmental disturbances. In this study, transcriptomics and label-free proteomics were combined to investigate Atlantic salmon (Salmo salar) sampled from three different field locations within the Baltic Sea (Baltic Main Basin (BMB), Gulf of Finland (GoF), and Bothnian Sea (BS)) during marine migration. The expression of several stress related mRNAs and proteins of xenobiotic metabolism, oxidative stress, DNA damage, and cell death were increased in salmon from GoF compared to salmon from BMB or BS. Respiratory electron chain and ATP synthesis related gene ontology-categories were upregulated in GoF salmon, whereas those associated with RNA processing and synthesis, translation, and protein folding decreased. Differences were seen also in metabolism and immune function related gene expression. Comparisons of the transcriptomic and proteomic profiles between salmon from GoF and salmon from BMB or BS suggest environmental stressors, especially exposure to contaminants, as a main explanation for differences. Salmon feeding in GoF are thus “disturbed by hazardous substances”. The results may also be applied in evaluating the conditions of pelagic ecosystems in the different parts of Baltic Sea.

Metabolic responses to temperature changes are crucial for maintaining the energy balance of an individual under seasonal temperature fluctuations. To understand how such responses differ in recently isolated populations (

Purpose A-type peptide, a natriuretic peptide belonging to the natriuretic peptide family, has been shown to be increased in the vitreous of patients suffering from diabetic retinopathy and that human retina has a well-developed natriuretic peptide system. The stimulus to which the synthesis of natriuretic peptides responded in these patients has, however, remained unknown. As the natriuretic peptides have recently been shown to respond to hypoxic conditions, the genes of both A-type and B-type have a hypoxia-response element (HRE) in their promoter sequence, we therefore hypothesized that hypoxia in the human retinal pigment epithelium will increase the secretion of NT-proBNP, the most common natriuretic peptide monitored in clinical medicine. Methods We used cultured human retinal pigment epithelium cell line (ARPE-19) which was exposed either to normoxia or to hypoxia for 2 hr, 4 hr, 6 hr and 24 hr. NT-proBNP was measured with enzyme immunoassay, VEGF with ELISA and HIF-1α with Western blotting. Results Hypoxia induced VEGF 165 release in culture medium and HIF-1α expression in cultured ARPE-19 cells. Time-dependent NT-proBNP release was detected when the ARPE-19 cells were cultured under normoxia. When hypoxia was induced, a statistically significant increase in NT-proBNP release was demonstrated in the culture medium. Conclusions Hypoxic conditions increase the release of a natriuretic peptide from retinal pigment epithelium (RPE) cells. The secretion of VEGF was also enhanced. The responses were associated with the up-regulation of the HIF-1α transcription factor. These results explain the previous findings from patients with diabetes, which also suggest that hypoxia is a ubiquitous stimulus for the secretion of natriuretic peptides in human body.

Oxidative stress has been suggested as a mediator in life-history trade-off. By spending more resources on for example reproduction an organism might sacrifice its antioxidant defence. So far, most ...

Our aim was to study the effects of hypoxia on the release of salmon cardiac peptide (sCP) from an isolated heart ventricle of trout during a constant mechanical load. Trout heart ventricles were studied in vitro. The ventricle was placed in an organ bath at 12 °C in which a constant mechanical load could be imposed on the ventricle while buffer solution was circulating. Ventricles were field-stimulated with a supramaximal voltage pulse at a rate of about 0.3 s−1. Samples of 1 ml were collected at an interval of 10 min for 200 min from the organ bath and assessed with a radioimmunoassay for sCP. After a control period of 20 min, ventricles were exposed to hypoxia produced with N2 gassing (n = 9) or to hypoxia with 20 mM BDM, a nonselective myosin ATPase inhibitor locking cross-bridges in a pre-power-stroke state inhibiting force production with normal electrical activity (n = 10). In this model and setup, hypoxia stimulated the release of sCP, but the interindividual variation in the response was large. At the end of hypoxia exposure, the concentration of sCP in the organ bath was about sixfold higher than at the start of the exposure (P

Antioxidant defense has an important role in the protection of organisms against oxidative stress caused by reactive oxygen species (ROS). Many metals are capable of generating ROS and inducing oxidative damage, and may therefore lead to changes in oxidative regulation. We studied species-specific variation in the oxidative status of great tit (Parus major), blue tit (Cyanistes caeruleus) and pied flycatcher (Ficedula hypoleuca) nestlings in a vicinity of a non-ferrous smelter. Non-enzymatic (glutathione [tGSH], GSH:GSSG ratio, and carotenoids) and enzymatic (glutathione peroxidase [GP], glutathione-S-transferase [GST], superoxide dismutase [SOD], and catalase [CAT]) antioxidants were evaluated to determine the effects of metal exposure on the oxidative status of the birds. We found strong evidence of interspecific variation in CAT and SOD activities, whereas less variation was observed in parameters related to glutathione metabolism. Oxidative state (in terms of tGSH and GSH:GSSG) did not vary between species, suggesting that different species may employ different antioxidant pathways to achieve the same oxidative state. Oxidative status was only weakly related to metal exposure, and these associations were further obscured by species-specific environmental effects. Our results indicate that effects on oxidative status observed in one species cannot be generalized to other ones. Future work should attempt to incorporate species-specific biology and environmental context into assessments of contaminant impacts on oxidative regulation of passerine birds.

Recent studies of long-distance migratory birds show that behavioural and physiological changes associated with predictable or unpredictable challenges during the annual cycle are distinctively regulated by hormones. Corticosterone is the primary energy regulating hormone in birds. Corticosterone levels are elevated during stresses but they are also modulated seasonally according to environmental conditions and life-history demands. We measured the baseline and stress-induced levels of corticosterone in the barn swallow (Hirundo rustica L.) just before spring and autumn migrations in South Africa and Finland, respectively. Barn swallows completing their pre-breeding moult had low body condition (residual body mass) and high baseline corticosterone levels in the wintering grounds. In contrast, baseline corticosterone levels in Finland were low and not related to residual mass. These data contradict the first prediction of the migration modulation hypothesis (MMH) by showing no association with baseline corticosterone levels and pre-migratory fuelling. Yet, the adrenocortical response to the capture and handling stress was notably blunted in South Africa compared to a strong response in Finland. Further, individuals that had started fuelling in Finland showed a reduced response to the handling stress. Taken together, elevated baseline corticosterone levels and high residual mass may blunt the adrenocortical response in long-distance migrants and aerial feeders such as the barn swallow. This observation lends support to the second prediction of the MMH.

The acute toxicity of organic tin compounds (OTCs) has been studied in detail. However, due to their complex nature, very little is known about species-specific methods of accumulation and consequences for food-webs. Chironomids, on which e.g. Daubenton's bats feed, may act as vectors for the transport of organic tin compounds from aquatic to terrestrial ecosystems. Bats are prone to environmental toxins because of their longevity and their ecological role as top predators. Organic tin compounds are associated with increased formation of reactive oxygen species and associated oxidative damage as well as suppression of immune function. The present paper investigates whether the OTC, tributyltin (TBT) and its metabolite, dibutyltin (DBT), accumulate in natural populations of Daubenton's bats and whether TBT-associated effects are seen in general body condition, redox balance, redox enzyme activities, associated oxidative damage of red blood cells and complement function. We discovered the concentration of bat fur DBT correlated with local marine sediment TBT concentrations. However, we did not find a correlation between the explanatory factors, bat fur DBT and marine sediment TBT concentrations, and several physiological and physical response variables apart from complement activity. Higher DBT concentrations resulted in weaker complement activity and thus a weaker immune response. Although the observed physiological effects in the present study were not strongly correlated to butyltin concentrations in fur or sediment, the result is unique for natural populations so far and raises interesting questions for future ecotoxicological studies.

In the present study, we surveyed developmental changes in the transcription of growth hormone (gh), insulin-like growth factor-I (igf-I), ghrelin (ghrl) and vascular endothelial growth factor (vegf) genes in the largest freshwater fish, European sturgeon (Beluga, Huso huso) and compared the same parameters to that of its phylogenically close moderate-sized species, Persian sturgeon (Acipenser persicus). The transcripts of gh, igf-I, ghrl and vegf were detected at all developmental time-points of Persian sturgeon and Beluga from embryos to juvenile fish. Changes in normalized gh, igf-I, ghrl and vegf transcription by using the geometric average of genes encoding ribosomal protein L6 (RPL6) and elongation factor (EF1A) over the time of development of Persian sturgeon and Beluga were statistically significant (P

In our previous study, for the first time in any fish species, the mRNA expression of L-Gulono-γ-lactone oxidase (GULO) was investigated during early development of Persian sturgeon, Acipenser persicus, and it was demonstrated that GULO mRNA was expressed throughout the development of Persian sturgeon. Accordingly, it was suggested that the initial production of GULO and probably ascorbate might occur before hatching during the embryonic stage. To evaluate the correspondence between transcript and protein level, GULO activity of Persian sturgeon during ontogenetic development of embryos and larvae was investigated. The GULO activity was detected in all time-points of the development of Persian sturgeon from embryos to the late larval stage indicating the start of ascorbate synthesis before hatching. However, there was a marked difference in the mRNA/protein activity ratio at different times. As the GULO mRNA and protein production peaks were observed during a time course from embryonic to early larval dev...

The heat shock response (HSR) refers to the rapid production of heat shock proteins (hsps) in response to a sudden increase in temperature. Its regulation by heat shock factors is a good example of how gene expression is transcriptionally regulated by environmental stresses. In contrast, little is known about post-transcriptional regulation of the response. The heat shock response is often used to characterize the temperature tolerance of species with the rationale that whenever the response sets on, a species is approaching its lethal temperature. It has commonly been considered that an increase in hsp mRNA gives an accurate indication that the same happens to the protein level, but this need not be the case. With climate change, understanding the effects of temperature on gene expression of especially polar organisms has become imperative to evaluate how both biodiversity and commercially important species respond, since temperature increases are expected to be largest in polar areas. Here we studied the HSR of two phylogenetically related Arctic species, which differ in their temperature tolerance with Arctic charr having lower maximally tolerated temperature than Atlantic salmon. Arctic charr acclimated to 15°C and exposed to 7°C temperature increase for 30 min showed both an increase in hsp70 mRNA and hsp70 whereas in salmon only hsp70 mRNA increased. Our results indicate that the temperature for transcriptional induction of hsp can be different from the one required for a measurable change in inducible hsp level. The species with lower temperature tolerance, Arctic charr, are experiencing temperature stress already at the higher acclimation temperature, 15°C, as their hsp70 mRNA and hsp70 levels were higher, and they grow less than fish at 8°C (whereas for salmon the opposite is true). Consequently, charr experience more drastic heat shock than salmon. Although further studies are needed to establish the temperature range and length of exposure where hsp mRNA and hsp level are disconnected, the observation suggests that by measuring both hsp mRNA and hsp level, one can evaluate if a species is approaching the higher end of its temperature tolerance, and thus evaluate the vulnerability of an organism to the challenges imposed by elevated water temperature.

DNA barcoding has become a useful tool in many contexts and has opened up a completely new avenue for taxonomy. DNA barcoding has its widest application in biodiversity and ecological research to detect and describe diversity whenever morphological discrimination is difficult or impossible (e.g., in the case of species lacking diagnostic characters, early life stages, or cryptic species). In this chapter, we outline the utility of including physiological parameters as part of species description in publicly available databases that catalog taxonomic information resulting from barcoding projects. Cryptic species or different life stages of a species often differ in their physiological traits. Thus, if physiological aspects were included in species definitions, the presently cryptic species could be distinguished. We furthermore give suggestions for physiological information that should be included in a species description and describe potential applications of DNA barcoding for research with physiological components.

DNA barcoding has become a useful tool in many contexts and has opened up a completely new avenue for taxonomy. DNA barcoding has its widest application in biodiversity and ecological research to detect and describe diversity whenever morphological discrimination is difficult or impossible (e.g., in the case of species lacking diagnostic characters, early life stages, or cryptic species). In this chapter, we outline the utility of including physiological parameters as part of species description in publicly available databases that catalog taxonomic information resulting from barcoding projects. Cryptic species or different life stages of a species often differ in their physiological traits. Thus, if physiological aspects were included in species definitions, the presently cryptic species could be distinguished. We furthermore give suggestions for physiological information that should be included in a species description and describe potential applications of DNA barcoding for research with physiological components.

The hormonal environment during early development, such as maternally derived androgens in bird eggs, shapes the development of the offspring in ways that may have important long-term consequences for phenotype and behavior and, ultimately, fitness. We studied the long-term effects of yolk androgens on several phenotypic and physiological traits in male and female pied flycatchers (Ficedula hypoleuca) by experimentally elevating yolk androgen levels and rearing birds in common-garden environment in captivity. We found that high yolk androgen levels increased the basal metabolic rates in both females and males in adulthood. High yolk androgen levels did not affect male melanin coloration or plumage ornaments, or timing or speed of moult in either sex. No effect of androgen treatment on cell-mediated or humoral immune response was found in either sex. Covariation among the measured phenotypic traits was further not altered by androgen treatment. Our results suggest that exposure to high androgen levels can have long-lasting effects on some offspring traits, but do not seem to lead to different phenotypes. Furthermore, the role of yolk androgens affecting sexually selected male traits in our study species seems to be minor. The fitness consequences of yolk androgen-induced higher metabolic rates remain to be studied.

Due to their adsorptive behavior, organotin compounds (OTCs), such as tributyltin (TBT), are accumulated in aquatic sediments. They resist biodegradation and, despite a ban in 2008, are a potential source for future exposure. Sediment OTCs have mostly been measured from sites of known high concentrations such as ports, shipping lanes, and marine dredging waste sites. The possible flow of OTCs from marine to terrestrial ecosystems, however, has not been studied. In the present study, the authors assessed whether sediments in common reed beds (Phragmites australis) accumulate TBT and whether chironomid (Diptera: Chironomidae) communities developing in reed-bed sediments act as vectors in the transfer of TBT from aquatic to terrestrial ecosystems in the Airisto channel, Archipelago Sea. The authors also investigated whether distance from the only known source and depth and TBT concentration of the adjacent shipping lane affect reed-bed concentrations. Thirty-six sites along the Airisto channel were sampled at 2-km intervals with triplicate samples from reed beds and the adjacent shipping lane for sediment and seven reed-bed sites for chironomids, and these were analyzed with an solid phase extraction liquid chromatography tamdem mass spectrometry method. The closer to the source the sample site was, the higher the measured TBT concentrations were; and the deeper the shipping lane, the lower the concentration of TBT in reed-bed sediments. The chironomid TBT concentrations correlated with reed-bed sediment TBT concentrations and showed evidence of accumulation. Therefore, TBT may be transferred, through the food web, from aquatic to terrestrial ecosystems relatively close to a source through ecosystem boundaries, such as common reed beds, which are areas of high insect biomass production in the Archipelago Sea. Environ. Toxicol. Chem. 2012; 31: 1781–1787. © 2012 SETAC

Sea surface microlayer (SML) and underlying subsurface water (SSW) bacterial communities from the Baltic Sea were studied using culturing, flow cytometry and terminal restriction fragment length polymorphism (T-RFLP). Dissolved organic matter (DOM) of water samples was determined using high performance liquid size-exclusion chromatography and by measuring dissolved organic carbon. Cadmium, copper, lead and mercury were also measured from the water samples. Two methods were compared for SML sampling: metal screen (MS) and polycarbonate membranes (PCM). All sampling sites were located in the Archipelago Sea off the southwest coast of Finland. The hypothesis was that because of its unique features, such as accumulation of nutrients and pollutants and being unshaded from UV-radiation, the SML would support a different bacterial community to that of SSW. T-RFLP profiles showed differences between the bacterial community compositions of SML and SSW at most sampling sites. In addition, temporal and sampling-site-dependent differences were observed. There were no significant differences in DOM in SML and SSW samples. In most cases, the number of bacteria was slightly higher in the SML than in SSW. No significant cytotoxicity was observed in any of the water samples. Since no temporal trends were obvious, our results suggest that, although bacterial communities in the SML Archipelago Sea differ to some extent from the ones living in SSW, SML is a dynamic environment where bacterial communities possibly develop originally from bacterioplankton and change over time.

Toxicological responses of an organism are disturbances of function. This as a starting point we review and discuss issues that we consider important in applying functional genomics to aquatic toxicology. Functional genomics includes all the steps in gene expression pathway. Thus, ultimately the goal is to relate genome information to protein activity. In ecotoxicogenomics the toxicological responses must further be combined with responses to natural environmental changes. We focus on fish, but also consider commonly used invertebrates, mainly Daphnia. We first go through the toxicologically important features of genomes of aquatic animals, and then review the reference gene approach to quantify transcript amount. Thereafter we emphasize the need to relate the mRNA and protein levels, and protein activity of individual genes. Finally we discuss how functional genomic investigations may be important in resolving current environmental problems and give our views of valuable future research topics.