France

A typical feature of many biological materials is the ability to react to experienced mechanical stress, as described in the “mechanostat” model by H.M. Frost. In this study we investigated so far unknown possible reactions to stress of one of the evolutionary most successful forms of skeletons: the insect exoskeleton. Locusts (Locusta migratoria) were raised under controlled conditions at increased mechanical load using a custom build centrifuge. Although the increase of mechanical stress notably decreased the survival rate, locusts surprisingly survived for several weeks at up to 8g. Biomechanical properties such as strength and stiffness of locust hindleg tibia were characterised using standardized mechanical tests. Further analysis using X-ray microtomography and a novel, simple staining method examine possible responses of the exoskeletons two main layers, exocuticle (outer layer) and endocuticle (inner layer). For the first time, our results show that insect exoskeleton cuticle material (e.g. Youngs modulus, bending strength) and structural properties (thickness of exo-, and endocuticle) were significantly influenced by the experienced mechanical stress. These findings not only allow us to better understand the biomechanical role of exo- and endocuticle within an insect exoskeleton however also help us to discuss the evolutionary context and validity of the “mechanostat” model for the largest and most successful group of animal species.

SEB Annual Conference 2022

Effect of mechanical stress on insect exo- and endocuticle

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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

technical paper

Evolutionary studies often search for population divergence, increasingly at microgeographic spatial scales and across contemporary time scales, reflecting the extent and pace of natural selection. The expectation tends to be that local populations should adapt to environmental variation, including variation associated with human-modified environments. A long history of theoretical and empirical studies supports this expectation, and indeed natural selection should diverge populations as it drives them toward local adaptive optima. Yet, perhaps more often than we are eager to admit, we fail to find evidence for local adaptation, and when this happens, we struggle to describe the mechanisms that can generate these patterns. For instance, what do we make of data that fail to clearly demonstrate local adaptation, such as when presumptively divergent populations are found to be equivalently adapted to contrasting environments? Or, how do we interpret the persistence of locally maladaptive trait variation? Or, how do we reconcile adaptive population demographic trajectories with maladaptive trait variation? Here, we discuss a framework of (mal)adaptation that emphasizes the importance of considering both absolute and relative fitness when interpreting local population divergence. We then use this framework to interpret outcomes from amphibian populations that show evidence of both adaptive and maladaptive divergence in response to road adjacency and runoff pollution.

Toward a unified approach to understanding (mal)adaptation

Blue-mussels from the M. edulis species-complex (M. edulis x M. trossulus x M. galloprovincialis) are an abundant element of the benthos community, found in the high latitude habitats of both hemispheres of the globe. Aside their ecological value, these foundation species are relevant to the aquaculture industry, with over 182 thousand tonnes produced globally each year. These taxa withstand a wide range of environmental conditions and the 3 species easily hybridise in regions where their distribution overlaps. Significant effort has been made to investigate the consequences of environmental stress on the physiology of these species. However, our understanding on the genomic mechanisms underlying hybridisation and local adaptation remains limited. To refine our understanding of such processes in this species complex, we sequenced 24 blue mussel populations distributed globally using a whole-genome low coverage approach. A total of 50k SNPs were used to develop a medium-density SNP-array, a tool that will allow the rapid and consistent genotyping of individuals. To guarantee that putative SNPs associated with environmental stress were included in the array, we selected mussel populations thriving across a gradient of environmental conditions in terms of temperature, salinity and CO2. The rapid high-throughput screening of SNPs facilitates the investigation of genomic structure among populations and can contribute to our understanding on ongoing local adaptation and hybridisation in these taxa. Besides, genome wide association studies, combining omics with physiology, will allow the characterisation of genotypes resilient to environmental stress, relevant in light of climate change.  

Exploring genomic diversity within the blue mussel species-complex

Social context influences all aspects of life, including how organisms interact with their surroundings, perceive stressors, and respond to toxicants in their environment. With the condition of aquatic environments declining worldwide, furthering our understanding of the factors that affect the success of species will be essential for their survivability. As such, we examined how copper (Cu) contamination affects behaviour and physiology of three-spined stickleback (Gasterosteus aculeatus) as a function of their social environment. We hypothesized that exposing sticklebacks to Cu, while experiencing the stress of isolation, will lead to greater uptake of Cu and subsequent behavioural responses, as opposed to exposing sticklebacks to Cu in a group setting. Wild-caught fish were exposed to environmentally relevant Cu concentrations of 50µg/L and 150µg/L for 96 hours then assessed for activity level, social cohesion, and foraging. Furthermore, tissues (gill, liver, and intestine) were collected to measure organ-specific bioaccumulation of ions and enzyme activity (Na+/K+/ATPase, H+-ATPase). The interplay between toxicology and social behaviour is not well understood, and as such is not considered in risk assessment standard practice. This potentially leaves a significant gap in our understanding of organismal response to toxicants. Observing response to copper after isolation and group exposures may highlight the need to account for the social environment to truly represent the vulnerability of populations.

Influences of Anthropogenic Stress on the Sociality of Three-Spined Stickleback (Gasterosteus Aculeatus)

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.

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

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.

Effect of mechanical stress on insect exo- and endocuticle

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