Cardiovascular oxygen transport and peripheral oxygen extraction capacity contribute to acute heat tolerance in European seabass.

This study evaluated whether different parameters describing cardiovascular function, energy metabolism, oxygen transport and oxidative stress were related to the critical thermal maximum (CTMAX) of European seabass (Dicentrarchus labrax) and if there were differential changes in these parameters during and after heat shock in animals with different CTMAX in order to characterize which physiological features make seabass vulnerable to heat waves. Seabass (n = 621) were tested for CTMAX and the physiological parameters were measured in individuals with good or poor temperature tolerance before and after a heat shock (change in temperature from 15 °C to 28 °C in 1.5 h). Fish with good thermal tolerance had larger ventricles with higher maximal heart rate during the heat shock than individuals with poor tolerance. Furthermore, they initially had a high ventricular Ca2+-ATPase activity, which was reduced to a similar level as in fish with poor tolerance following heat shock. The activity of heart lactate dehydrogenase increased in fish with high tolerance, when they were exposed to heat shock, while the aerobic enzyme activity did not differ between groups. The tolerant individuals had smaller red muscle fibers with higher myoglobin content than the poorly tolerant ones. The poorly tolerant individuals had higher hematocrit, which increased with heat shock in both groups. The poorly tolerant individuals had also higher activity of enzymes related to oxidative stress especially after heat shock. In general, CTMAX was not depending on merely one physiological factor but several organ and cellular parameters were related to the CTMAX of seabass and when working in combination they might protect the highly tolerant seabass from future heat waves.

Functional Genomics of Fish Erythrocytes.

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.

Copper exposure improves the upper thermal tolerance in a sex-specific manner, irrespective of fish thermal history.

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.

Environmentally driven changes in Baltic salmon oxidative status during marine migration.

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.

Hsp70s transcription-translation relationship depends on the heat shock temperature in zebrafish.

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.

Environmental regulation of the function of circulating erythrocytes via changes in age distribution in teleost fish: Possible mechanisms and significance.

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.

Regulation of erythrocyte function: Multiple evolutionary solutions for respiratory gas transport and its regulation in fish.

Gas transport concepts in vertebrates have naturally been formulated based on human blood. However, the first vertebrates were aquatic, and fish and tetrapods diverged hundreds of millions years ago. Water-breathing vertebrates live in an environment with low and variable O2 levels, making environmental O2 an important evolutionary selection pressure in fishes, and various features of their gas transport differ from humans. Erythrocyte function in fish is of current interest, because current environmental changes affect gas transport, and because especially zebrafish is used as a model in biomedical studies, making it important to understand the differences in gas transport between fish and mammals to be able to carry out meaningful studies. Of the close to thirty thousand fish species, teleosts are the most species-numerous group. However, two additional radiations are discussed: agnathans and elasmobranchs. The gas transport by elasmobranchs may be closest to the ancestors of tetrapods. The major difference in their haemoglobin (Hb) function to humans is their high urea tolerance. Agnathans differ from other vertebrates by having Hbs, where cooperativity is achieved by monomer-oligomer equilibria. Their erythrocytes also lack the anion exchange pathway with profound effects on CO2 transport. Teleosts are characterized by highly pH sensitive Hbs, which can fail to become fully O2 -saturated at low pH. An adrenergically stimulated Na+ /H+ exchanger has evolved in their erythrocyte membrane, and plasma-accessible carbonic anhydrase can be differentially distributed among their tissues. Together, and differing from other vertebrates, these features can maximize O2 unloading in muscle while ensuring O2 loading in gills.

Water-soluble fraction of crude oil affects variability and has transgenerational effects in Daphnia magna.

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.

Individual variation in aquatic toxicology: Not only unwanted noise.

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.

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls.

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.

Circadian rhythms and environmental disturbances - underexplored interactions.

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.

Cold temperature represses daily rhythms in the liver transcriptome of a stenothermal teleost under decreasing day length.

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 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 and 8°C) for 1 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 with strong daily variation in warm-acclimated fish, suggesting that 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 with 8°C. The results point out mechanisms that can affect the ability of fish to adapt to increasing temperatures caused by climate change.

Systemic Hypoxia Increases Circulating Concentration of Apelin in Humans.

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. BACKGROUND: 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. METHODS: Ten healthy young men (age 29 ± 5 years, body mass index 24.7 ± 2.8 kg/m2) breathed normobaric hypoxic gas mixture (11% O2/89% N2) for 1 hour. Venous blood samples were obtained immediately before, and 2 and 24 hours after the start of the hypoxic exposure and analyzed for circulating apelin concentrations. RESULTS: Arterial 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). CONCLUSION: In conclusion, in line with cell culture and animal studies, acute systemic hypoxia increases circulating apelin concentration also in humans.

Influence of crude oil exposure on cardiac function and thermal tolerance of juvenile rainbow trout and European sea bass.

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.

In vitro study on the regulation of cellular mRNA levels by changes in transcription rate and transcript stability in fish red blood cells.

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 ß-adrenergic agonist isoproterenol). We found that ß-adrenergic stimulation increases the stability of the ß3b-adrenergic receptor (ß3b-ar) mRNA, suggesting that mRNA stability can play a role in the regulation of hormonal stress responses in fish. The ß3b-ar gene encodes a unique ß-adrenergic receptor subtype in fish red blood cells which controls the ß-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 ß-adrenergic receptor response. After ß-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 ß-AR is fundamental to the molecular and physiological responses to hypoxia and that Hif1a might have additional functions than those already known.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus.

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats.

Transcriptomic Analysis of Young and Old Erythrocytes of Fish.

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.

Different Relationship between hsp70 mRNA and hsp70 Levels in the Heat Shock Response of Two Salmonids with Dissimilar Temperature Preference.

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.