France

Sources of metals in the Antarctic marine environment can vary due to glacial melt. The limpet Nacella conccina, a well-distributed specie at Antarctic coastal zone, is able to deal with some different copper concentrations. During the experiments limpets were exposed to 0.12 µgL-1 and 0.25 µgL-1 of copper at 12, 24, 48 h (N=6/metal/concentration/time, temperature 0°C and salinity 34 ppt). Gills were analysed using proteomics techniques. We detected 78 significantly altered proteins (two-way ANOVA, p&amp;lt;0.02) on 2D electrophoresis gels. Among these 47 were identified using tandem mass spectrometry. Antioxidant proteins including Mn-superoxide dismutase and ferritin were identified. This is an indication of the increased production of ROS. The chaperones involved in the maturation of proteins present in the endoplasmic reticulum showed variations in function of a possible change in the cellular redox state. Modifications in the acid-base balance of gill cells can be linked to changes in carbonic anhydrase after exposure to copper. The dynamics of proteins such as arginine kinase, glycolytic triose phosphate isomerase, as well as pyruvate dehydrogenase, can be linked to a higher rate of ATP renewal. We hypothesised that substituting protein-binding metals by copper causes an increase in ROS and acid-base effects in the gills, leading to protein maturation inhibition in the endoplasmic reticulum, an increment in ATP buffering by arginine kinase and glycolytic proteins, and changes in Cdc42 abundance impacting the cytoskeletal filaments. These observations help to shed light on the interrelationships between metabolism, energy balance and short time copper stress tolerance in Antarctic limpets.

SEB Annual Conference 2022

Antarctic limpet cell physiology scope after copper exposition: a proteomic approach

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

Sources of metals in the Antarctic marine environment can vary due to glacial melt. The limpet Nacella conccina, a well-distributed specie at Antarctic coastal zone, is able to deal with some different copper concentrations. During the experiments limpets were exposed to 0.12 µgL-1 and 0.25 µgL-1 of copper at 12, 24, 48 h (N=6/metal/concentration/time, temperature 0°C and salinity 34 ppt). Gills were analysed using proteomics techniques. We detected 78 significantly altered proteins (two-way ANOVA, p&lt;0.02) on 2D electrophoresis gels. Among these 47 were identified using tandem mass spectrometry. Antioxidant proteins including Mn-superoxide dismutase and ferritin were identified. This is an indication of the increased production of ROS. The chaperones involved in the maturation of proteins present in the endoplasmic reticulum showed variations in function of a possible change in the cellular redox state. Modifications in the acid-base balance of gill cells can be linked to changes in carbonic anhydrase after exposure to copper. The dynamics of proteins such as arginine kinase, glycolytic triose phosphate isomerase, as well as pyruvate dehydrogenase, can be linked to a higher rate of ATP renewal. We hypothesised that substituting protein-binding metals by copper causes an increase in ROS and acid-base effects in the gills, leading to protein maturation inhibition in the endoplasmic reticulum, an increment in ATP buffering by arginine kinase and glycolytic proteins, and changes in Cdc42 abundance impacting the cytoskeletal filaments. These observations help to shed light on the interrelationships between metabolism, energy balance and short time copper stress tolerance in Antarctic limpets.

technical paper

The ability of populations to expand their geographic ranges, whether as invaders or climate migrants, presents among the most serious global problems today. However, fundamental mechanisms that enable certain populations to rapidly adapt to novel habitats remain poorly understood. In recent years, populations of the copepod Eurytemora affinis species complex have invaded freshwater habitats multiple times independently from saline sources. These copepods are dominant grazers in aquatic habitats throughout the Northern Hemisphere and support major fisheries. Intriguingly, evolutionary changes during these salinity transitions often involved the same loci, with selection often acting on the same SNPs (alleles), in wild populations and laboratory selection lines. In our laboratory natural selection experiment, the same SNPs were favored across replicate selection lines far beyond expectations. In both wild populations and laboratory selection lines, ion transporter genes appeared as the dominant functional category under selection. Using extensive simulations, we found that this degree of parallelism was consistent with synergistic epistasis among alleles responding in concert across replicate selection lines for this polygenic trait (salinity tolerance). The effect of synergistic epistasis appeared greater than selection from balanced polymorphisms in promoting parallel evolution. Our results were consistent with mechanisms of ion uptake from dilute habitats, requiring the coordinated action of cooperating ion transporter proteins. Most notably, our results suggest that a very specific set of ion transporter alleles might be necessary for freshwater adaptation to occur in this system, pointing to a canalized evolutionary pathway. Thus, we find strong support for a novel and potentially widespread mechanism, namely positive epistasis, in promoting this parallel and canalized response. This research is funded by NSF-OCE, NSF-DEB, and ANR (Macron’s Make Our Planet Great Again award).

Mechanisms of parallel adaptation in a widespread copepod

Acute environmental stresses including high temperature can lead to the breakdown of the mutualism between reef-building corals and their symbiotic algal partners, ultimately leading to bleaching. However, variation in the thermal tolerance of different coral-algal symbioses has been described repeatedly, suggesting differences in sensitivity to heat among the partners. Since high temperature perturbs the structure and function of macromolecules, these differences in whole-organism temperature sensitivity may occur in part because of differences in the thermal stability of such molecules, including proteins. Here, we examine temperature sensitivity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in three coral species – Acropora millepora, Orbicella faveolata, and Astrangia poculata – and three genera of Symbiodiniaceae – Breviolum, Cladocopium, and Durusdinium – to explore whether differences exist between coral and algal orthologs in stability and function at high temperatures. In addition, we examine whether differences in thermal sensitivity exist within GAPDHs of Symbiodiniaceae taxa that have been suggested to possess different levels of thermal tolerance. We use recombinant GAPDH to examine both thermal stability (melting temperature measured via differential scanning fluorimetry) and functional measures of thermal sensitivity (Michaelis constant Km, activation energy Ea) in each ortholog. Our data, complemented by molecular dynamics simulations, indicate that GAPDHs in coral are less sensitive to high temperature perturbation than are algal orthologs, both in kinetics and stability. These results suggest that in some coral-algal symbioses, certain enzymes of the dinoflagellate are more sensitive to heat stress than the host, which may contribute to derangement of the symbiosis and resultant bleaching.

Differential sensitivity to high temperature in glyceraldehyde-3-phosphate dehydrogenases of coral and algal symbionts

Giant clams are threatened by overexploitation for human consumption, their valuable shells and the aquarium trade. Consequently, these iconic megafauna are extinct in some former areas of their range and included in the IUCN Red List of Threatened Species and CITES list. Now, giant clams are also threatened by rapid environmental change from both a suite of local- to regional-scale stressors, such poor water quality and pollution, and global change stressors, including ocean heating and acidification. The interplay between local- to regional-scale and global-scale drivers is likely to cause an array of lethal and sub-lethal effects, potentially limiting giant clam depth distribution on coral reefs and decreasing suitable habitat area within natural ranges. International efforts urgently need to reduce CO2 emissions to avoid lethal and sub-lethal effects on giant clams. Meanwhile, knowledge of ecological and physiological responses to local–regional and global stressors could play a critical role in the conservation of these species through rapid environmental change. Further work on how biological responses translate into habitat requirements as global change progresses, selective breeding for resilience, the capacity for rapid adaptive responses of the giant clam holobiont, and valuing tourism potential may help improve the prospects of these charismatic megafauna over coming decades.

Conserving threatened coastal wildlife during rapid environmental change: using biological responses to inform management strategies of giant clams

Insect species represent more than 70% of all animal species and are also known to have the highest metabolic rates and the most rapidly contracting muscles in nature. They have also colonized just about every habitat on earth and are thus confronted to a large range of temperature changes. We hypothesized that the capacity of insects’ mitochondria to oxidize different metabolic fuels is paramount for thermal adaptation in insects. By measuring oxygen consumption in permeabilized flight muscles of three different species (Apis mellifera, Drosophila melanogaster and Leptinotarsa decemlineata) from 6 to 45° C, we detected important and species-specific mitochondrial flexibility for substrate oxidation to sustain high oxygen consumption levels at high temperatures. Specifically, mitochondrial respiration sustained by complex I substrates sharply declined at high temperatures in A. mellifera and D. melanogaster, but this was compensated with important increases of succinate and/or glycerol-3-phosphate (G3P) oxidation. We then monitored for 12 months North American colonies of honeybees enduring extremely low and high temperatures, to assess the metabolism of honeybees transitioning from a summer to a winter phenotype. Our results show that complex I-induced respiration during winter is drastically reduced compared to summer, but that overall, mitochondrial respiration is increased during winter owing to succinate and G3P being prominent substrates used to keep the hive warm. Overall, our results suggests previously unknown adaptive mechanisms of mitochondria to temperature, and shed light on potential thermoregulating strategies used by insects' thorax muscles.

The versatility of mitochondrial substrate oxidation in insects: a keystone for temperature adaptation?

Hypoxia is a major stressor for aquatic organisms. In organisms like the intertidal oyster Crassostrea gigas, adaptations to frequent fluctuations in oxygen levels require metabolic plasticity to maintain mitochondrial function and adjust to shifts in substrate availability. This study investigated the effects of acute and long term hypoxia-reoxygenation (H/R) stress (15 min and 90 min at ~0% O2 for acute and long term exposures, respectively, and 10 min reoxygenation) on respiration (Mo2) and reactive oxygen species (ROS) generation in the gill and digestive gland mitochondria of C. gigas respiring on different substrates (pyruvate, succinate, and palmitate). Oyster mitochondria showed high capacity for succinate oxidation under normoxia in both studied tissues. Mitochondrial responses to H/R stress strongly depended on the oxidized substrate, the activity state of mitochondria and the type of hypoxia exposure. In both tissues, oxidizing exposure to both H/R stress suppressed Mo2 and ROS generation in the resting (LEAK state) mitochondria oxidizing NADH-linked substrates (palmitate/pyruvate). However, after acute H/R stress, mitochondria in ADP-stimulated (OXPHOS) state increased ROS production despite suppressed oxygen consumption. In mitochondria oxidizing FADH2–linked substrate (succinate), acute H/R exposure strongly increased Mo2 and ROS in LEAK and OXPHOS states, whereas; ROS production after long term H/R exposure was similar to normoxic conditions despite Mo2 suppression. Suppressed ability to oxidize NADH-linked substrates indicates that mitochondrial Complex I might be the key target of H/R induced injury in oyster mitochondria, which can be partially offset by robust succinate oxidation capacity to support ATP production during frequent oxygen fluctuations.

Tissue and substrate-dependent mitochondrial responses to Hypoxia/Reoxygenation stress in marine bivalve Crassostrea gigas (Thunberg, 1793).

Mitochondrial aerobic metabolism is suggested to play a central role in ecology and evolution by influencing animal performance, as well as acclimation and adaptation to environmental conditions through its impact on ATP, ROS and heat production. While mitochondrial aerobic metabolism is highly plastic, recent evidence suggest that consistent inter-individual differences through time do exist. Understanding the origin (e.g. genetic differences, early-life programming) of these consistent inter-individual differences is a key step to further understand how mitochondrial aerobic metabolism is shaping animal’s life and performance. We explored this question in a wild bird species (i.e. the pied flycatcher) by experimentally manipulating egg levels of thyroid hormones (i.e. prenatal environment), switching half of the chicks between nests after hatching (i.e. partial cross-fostering) and manipulating postnatal ambient temperature. By measuring mitochondrial aerobic metabolism of blood cells, we were able to show that the prenatal hormonal environment only had mild and sex-specific effects on mitochondrial aerobic metabolism. While variation in mitochondrial traits was only weakly explained by the nest of origin (i.e. genetic + prenatal effects: 10.8  2.4%), the postnatal environment (i.e. nest of rearing) explained a large amount of variance (41.6  6.2%). Yet, postnatal nest cooling had no clear effect on mitochondrial aerobic metabolism, thereby suggesting that other postnatal environmental factors such as food availability or quality must be more important. Our results highlight that while genetic and prenatal factors likely contribute to inter-individual variation in mitochondrial aerobic metabolism, postnatal environmental factors are probably its main drivers.

Half-opening the mitochondrial black box: genetic, prenatal and postnatal environmental determinants of mitochondrial aerobic metabolism in the wild

Temperature is among the most important factors shaping the biogeographical distribution of ectotherm species. Thermal constraints on aerobic metabolism have been observed in several phyla at both high and low temperature and thermal adaptations of mitochondrial function are believed to play an important role for the variable thermal performance of ectothermic animals. Using a comparative system of Drosophila species with different thermal tolerance, we examined the thermal sensitivity of mitochondrial function at both high and low temperature extremes to explore a potential relation to organismal thermal tolerance. Mitochondrial function was measured in permeabilised thoraces using a substrate-uncoupler-inhibitor-titration protocol to sequentially stimulate components of the electron transport system. Preliminary results show that complex-I-driven respiration is severely challenged at both high and low temperature. Specifically, complex-I-driven respiration fails at a species-specific temperature that correlates strongly with minimal and maximal tolerance (CTmin and CTmax). However, for all species and at all temperatures, it was possible to restore a stable total oxygen consumption across temperatures when alternative substrates were provided (proline, succinate and glycerol-3-phosphate). These observations suggest that loss of neuromuscular function and mortality at temperature extremes are more associated with failure of complex-I-driven respiration, rather than rates of fully stimulated respiration. Ongoing investigations are now examining if/how failure of complex-I-driven respiration could be associated with lowered energetic P:O ratios and/or increased production of reactive oxygen species, as these responses could explain the apparent connection to organismal failure.

Mitochondrial (dys)function at high and low temperature extremes – complex I as a common culprit?

Mitochondria play a key role in energy metabolism through cellular respiration. Despite their importance, they are uniquely exposed to perturbation because their functions depend on the correct interaction between two distinct genomes, the mitochondrial and the nuclear DNA. Even mild incompatibilities between the two genomes could impact mitochondrial functions with downstream repercussions on individual fitness. Climate change predictions estimate an increase in temperature and its variability, changes in food web structures, but also in the distribution of populations. Events that may generate mitonuclear mismatches (e.g. hybridization between separate populations) are therefore expected to increase in frequency following the shifts in thermal niches. In addition, temperature and dietary regimes are well-known metabolic stressors whose variation can potentially exacerbate mitonuclear incompatibilities. The aim of this research was to test how far mitonuclear interactions contribute to thermal and dietary adaptation or breakdown in changing environments. I employed experimental lines of the fruitfly Drosophila melanogaster, characterized by mitonuclear match or mismatch, to investigate the combined impact of mitonuclear genotype, temperature, and diet modulation over a wide array of phenotypic traits, including mitochondrial functions, reactive oxygen species metabolism and life-history trade-offs. Results suggest that mitonuclear interactions might impact organismal fitness in an unpredictable way, potentially influencing local adaptation in a mutating world.

The role of mitonuclear interactions in thermal and dietary adaptation

Niclosamide (Nic) and TFM (3-trifluoromethyl-4-nitrophenol) are used to control invasive sea lamprey populations in the Laurentian Great Lakes. While TFM is selective to sea lamprey, Nic is more toxic in lamprey and other species. Nic is co-applied (1%) with TFM, reducing TFM requirements without comprising selectivity. It is established that TFM uncouples mitochondrial oxidative phosphorylation in fish, but the mode of action of Nic in fishes remains unclear. Using isolated mitochondria from rainbow trout and upstream migrant sea lamprey livers, we tested the hypothesis that Nic uncouples oxidative phosphorylation when applied alone and in TFM:Nic mixtures. At low (&lt;1 uM) Nic concentrations, ADP-independent respiration was higher than controls and TFM alone, suggesting that Nic is a potent uncoupler in both species. At levels &gt;1 uM Nic, respiration rates collapsed in lamprey, but not in trout, suggesting that Nic has a different mode of action in the two species. In mixtures, the interaction between the lampricides was complex: in trout, lower doses of the mixture were less toxic than Nic alone, while in lamprey, the mixture was more toxic to the mitochondria than either lampricide alone. Taken together, our data show that Nic (i) uncouples oxidative phosphorylation, (ii) interacts differently with the trout and lamprey mitochondria and (iii) in TFM mixtures, it may increase specificity for the lamprey. Information on the mode of action of Nic and TFM:Nic mixtures will help identify the cellular targets of the lampricides, to better understand their interaction and prevent non-target effects. 

The complex mode of action of the lampricide niclosamide in isolated liver mitochondria

Locomotion is a key aspect associated with ecologically relevant tasks for
many organisms, therefore, survival often depends on their ability to perform
well at these tasks. Despite this significance, we have little idea how
different performance tasks are weighted when increased performance in
one task comes at the cost of decreased performance in another. Additionally,
the ability for natural systems to become optimized to perform a
specific task can be limited by structural, historic or functional constraints.
Climbing lizards provide a good example of these constraints as climbing
ability likely requires the optimization of tasks which may conflict with
one another such as increasing speed, avoiding falls and reducing the cost
of transport (COT). Understanding how modifications to the lizard bauplan
can influence these tasks may allow us to understand the relative weighting
of different performance objectives among species. Here, we reconstruct
multiple performance landscapes of climbing locomotion using a 10 d.f.
robot based upon the lizard bauplan, including an actuated spine, shoulders
and feet, the latter which interlock with the surface via claws. This design
allows us to independently vary speed, foot angles and range of motion
(ROM), while simultaneously collecting data on climbed distance, stability
and efficiency. We first demonstrate a trade-off between speed and stability,
with high speeds resulting in decreased stability and low speeds an
increased COT. By varying foot orientation of fore- and hindfeet independently,
we found geckos converge on a narrow optimum of foot angles
(fore 20°, hind 100°) for both speed and stability, but avoid a secondary
wider optimum (fore −20°, hind −50°) highlighting a possible constraint.
Modifying the spine and limb ROM revealed a gradient in performance.
Evolutionary modifications in movement among extant species over time
appear to follow this gradient towards areas which promote speed
and efficiency.

Antarctic limpet cell physiology scope after copper exposition: a proteomic approach

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Mechanisms of parallel adaptation in a widespread copepod

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The versatility of mitochondrial substrate oxidation in insects: a keystone for temperature adaptation?

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