France

Although there is much controversy regarding the effects of ocean acidification on the behaviour of marine fish, marine invertebrates have been much less studied. Dungeness crabs, Metacarcinus magister, are found on the West coast of North American and their commercial and recreational fishery is one of the most valuable fishery in the Pacific region, with annual revenues of $220 million. Dungeness crabs live in the deep-sea and enter intertidal areas in order to find food. Previous studies have found that they routinely encounter pCO2 concentrations of up to 1300 µatm in their coastal habitat and that these levels of ocean acidification can affect larval development and mechanoreceptors. We exposed adult Dungeness crabs to control (600 µatm) and high pCO2 (1300 µatm) for 10 days and assessed their behaviour and physiology using electrophysiology, gene expression, immunohistochemistry and transmission electron microscopy of their olfactory organs, the antennules. Dungeness crabs exposed to high pCO2 had a 25% lower rate of antennular flicking rate in response to all concentrations of cadaverine, a food cue, than crabs exposed to control pCO2. No differences in antennular flicking were found in response to ammonium chloride, a deterant, however there was a large increase in the expression of Rh proteins that function as ammonia transporters in their antennules indicating a potential compensatory mechanism. We propose that a more holistic approach to the overall question of the effects of ocean acidification needs to be taken at multiple levels of biological organization and using environmentally relevant odorants and concentrations.

SEB Annual Conference 2022

The effects of simulated ocean acidification on the behaviour and physiology of the economically important Dungeness crab, Metacarcinus magister

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.

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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

A central prediction of recent climate change models is that there will be an increase in thermal unpredictability and heatwave events. Sessile intertidal organisms experience a high degree of thermal unpredictability due to combined effects of solar radiation and tidal movement. How thermal unpredictability operates in microhabitats with different media, such as tidepool (aquatic) or tidally exposed (aerial) environments is poorly understood, especially when coupled with other important determinants of performance, such as food availability. Understanding how these factors interact to influence performance is essential towards understanding the effects of climate change on intertidal communities. We examined how acclimation to different levels of thermal predictability (no, predictable, unpredictable) with high and low food availability shape performance during a three-day heatwave in Mytilus californianus in two microhabitat treatments: tidepool and tidally exposed. During the heatwave, mussels were warmed to 35C each daytime low tide in their respective medium (water or air). Tissues samples were taken for biochemical analyses the day before, each day during, and the day after the heatwave. We found that microhabitat was the strongest driving factor for shaping physiological performance in Mytilus californianus. Food availability played a larger role in shaping performance in tidally exposed mussels, and the influence of thermal predictability depended on heatwave day. Our results suggest that the predicted increases in heatwave events and thermal unpredictability from climate change will not have a uniform effect in different microhabitats, which could have severe consequences for intertidal community and composition.

Heatwaves in the intertidal: how microhabitat, thermal unpredictability, and food availability shape physiological performance in mussels

Fluid mechanics is an important tool for understanding the biology and ecology of marine life. A major challenge in studying how microscopic aquatic organisms function in their natural habitats is integrating the different scales at which critical physical and biological processes occur. How can we make large-scale field measurements and models that include the behaviors and physical features of real organisms? Conversely, how can we design small-scale experiments to measure those biological factors under hydrodynamic conditions that reflect what the organisms actually experience in the large- scale ocean? I will discuss examples of some of the approaches we have used to span different scales in our studies of how the interaction between the locomotion of microscopic organisms and the turbulent, wave-driven water flow around them determines how they move through the environment. We studied how the microscopic larvae of bottom-dwelling marine animals navigate to suitable habitats on the sea floor by combining field flow measurements in marine environments with flume studies, experiments in fluidic devices, experiments with dynamically-scaled physical models, and agent-based models of different locomotory strategies in measured turbulent flow fields. 

Locomoting in a turbulent environment: Ways to study microscale processes in a large-scale ocean

Dynamic soaring harvests energy from a spatiotemporal wind gradient, allowing albatrosses to glide over vast distances. However, its use is challenging to demonstrate empirically, and has yet to be confirmed in other seabirds. We investigate how flap-gliding Manx Shearwaters optimize their flight for dynamic soaring. We do so by deriving a new metric, the horizontal wind effectiveness, that quantifies how effectively flight harvests energy from a shear layer. We evaluate this metric empirically for fine-scale trajectories reconstructed from bird-borne video data using a simplified flight dynamics model. We find that the birds’ undulations are phased with their horizontal turning to optimize energy harvesting. We also assess the opportunity for energy harvesting in long-range, GPS-logged foraging trajectories, and find that Manx Shearwaters optimize their flight to increase the opportunity for dynamic soaring during favourable wind conditions. Our results show how small-scale dynamic soaring impacts large-scale Manx Shearwater distribution at sea.




Optimisation of dynamic soaring in a flap-gliding seabird impacts its distribution at sea

Plants grow continuously throughout their life cycle. Plant cells within meristems- the actively growing regions at the tips of shoots and roots- undergo repeated division cycles to provide the cells needed for growth.  Division must be coordinated with cell growth because cells within a meristem maintain their average size when conditions are constant. However in response to changes in environmental conditions or in different genotype backgrounds, cell size is reset to a new average. Using combined modeling and imaging approaches, we have been studying the relationship between cell size control and cell cycle progression and we find evidence for two cell size control points within the Arabidopsis cell cycle at both the G1/S phase and G2/M phase boundaries, and that the adaptation of cell size to changing conditions appears to be an emergent property of the overall system of cell cycle control.

Decisions, decisions, decisions: Cell division and cell size control in Arabidopsis.

Spatial transcriptomics is a new technology for capturing and mapping whole transcriptome gene expression on histological sections close to single-cell resolution. This technique allows researchers to gain insight into organs’ molecular morphology, as well as regional changes in gene expression during disease progression, development, and evolution. We apply this technique to the light-absorbing retina within the developing mouse eye. The mouse retina lacks internal blood vessels at birth but develops these structures over the first four weeks, which changes the diffusion dynamics for O2 and nutrients and affects the metabolic phenotypes of the retinal cells. Using spatial transcriptomics, we have generated spatial molecular atlases of the retina and extracted combined information on nutrient diffusion distances and whole transcriptome gene expression. With this technique, we can now provide a new quantitative understanding of the cellular responses to changes in O2 and nutrient availabilities during organ development. 

Tracing the development of the eye using spatial transcriptomics

We have developed a super resolution cryo-fluorescence optical microscope in order to perform correlative imaging with the cryo Soft X-ray Tomography (SXT) beamline at the Diamond Light Source. Using Structured Illumination Microscopy (SIM) the microscope generates images at almost twice the achievable conventional optical resolution in 3 dimensions. By reducing the gap between the resolution in optical fluorescence images and the X-ray tomography significantly more information can be extracted from the correlated image sets. However, there are many barriers to reliably produce high quality fluorescence images of 3D samples in cryo while preserving the sample integrity. The sample must be kept cold, condensation must be avoided on both the sample and the optics, sample position must be highly stable and light must be collected at as high a numerical aperture as possible. We have open source control software providing a simple interface with high speed control of the wide range of hardware involved as well as a high degree of flexibility to provide a range of experimental options. Despite, these challenges we have been able to reliably produce images with sub 200 nm resolution and achieve correlative alignment at sub 100 nm. The CryoSIM and cryoSXT instruments are available for open access via peer revied applications.

CryoSIM: the trials and tribulations of correlative super resolution cryo fluorescence imaging.

Bisphenols enter the environment from the production and degradation of plastics, and are ubiquitous pollutants with endocrine disrupting effects. Thyroid hormones are key regulators of vertebrate metabolism and muscle function, and represent one of many endocrine targets disrupted by bisphenol S (BPS). Our aim was to test whether BPS disrupts locomotor performance in zebrafish in a thyroid-dependent manner. First, we established that BPS at an environmentally relevant concentration impaired swimming performance (Ucrit). We then conducted two subsequent but separate, fully factorial experiments using BPS exposure (present and absent) and induced hypothyroidism (hypo- and normo- thyroid) as factors to assess mitochondrial function and muscle protein composition as potential mechanisms by which BPS could disrupt locomotor performance. Hypothyroidism but not BPS exposure reduced maximal substrate oxidation and proton leak. Metrics of mitochondrial efficiency were not impact by BPS exposure but were altered by both temperature and hypothyroidism. BPS exposure decreased AMPK activity (pAMPK/AMPKtotal) in skeletal muscle but increased fast myosin heavy chain (MHC) proteins. BPS exposure did not strongly impact slow MHC or myosin enhancer factor 2 (MEF2). Hypothyroidism decreased AMPK activity, and increased fast and slow MHC and MEF2 so that BPS and hypothyroidism affected muscle in the same direction. Since AMPK activity is important to sustained exercise, the BPS-induced decrease in AMPK activity could explain the observed decrease in swimming performance.

Bisphenol S Exposure reduces locomotor function by affecting muscle function but not mitochondria

First identified as sexual hormones, estrogens were shown to have a key role in the development of gonads but also to be involved in the skeleton development and homeostasis. In mammals, cytosolic estrogen receptors (ERa and ERb) are expressed in osteoblasts, osteoclasts, osteocytes as well as chondrocytes and chondroblasts, regulating their activity to build up and maintain cartilage and bone. 
To expand our knowledge of estrogen signaling in skeletal development outside of mammals, we used the European sea bass Dicentrarchus labrax, to test the impact of two concentrations of the natural estrogen 17b estradiol (E2, 0.4 and 40 ng/L) on 6-to-23-day old post-hatching larvae. 
We evaluated the effects of E2 on the morphological characteristics of the larvae as well as on the mineralization of the cranium, vertebrae and fins using alizarin red staining. In addition, qPCR measurements were performed to analyze the expression of various genes known to be involved in the formation of cartilage and bone in vertebrates. We showed that the effect of estrogens varied depending on the developmental stage of the larvae and the state of mineralization of their skeleton at the time of treatment, and that these effects were dose-dependent.











Estradiol effects on early skeletogenesis in the European sea bass Dicentrarchus labrax

Anthropogenic changes in the environment are ubiquitos and affect organisms in many ways. Flexibility of the endocrine system may help organisms to response to those changes. However, disruption in the female endocrine function may lead to altered transfer of hormones in eggs or embryos, ie. transgenerational endocrine disruption, which can have long-lasting effects on offspring. Maternally derived thyroid hormones (THs) are essential for development, but poorly studied, especially in wild birds. Here, we provide three case examples of studies on transgenerational TH disruption. First, we studied how parental exposure to metal pollution, using repeated sampling of polluted and control areas across Europe, influenced egg TH levels in a passerine bird, the great tit. Second, we studied how long-term experimental parental herbicide exposure influenced egg THs and offspring traits in Japanese quails. Third, we studied experimetally how limitation of iodine, a key element of the TH molecule, influenced parental and egg THs in rock pigeons. We found no overall effect of parental metal exposure on egg THs, but the effect was dependent on calcium availability. Parental herbicide exposure did not influence egg THs, but affected hatchability and embryo traits. Parental iodine availability did not influence parental or egg TH levels, but decreased egg laying rate. Results of these case examples suggest that transgenerational endocrine disruption is a complex phenomena and should be further studied, as such legacy effects can delay adapting to environmental changes.

Transgenerational endocrine disruption - a potential mechanism for legacy effects of anthropogenic disturbance

Nocturnin (NOC) is a phosphatase involved in the transformation of NADP(H) to NAD(H), performing a key role in the regulation of metabolism. In mammals, it has been purposed that nocturnin expression is induced by E-box element activation. However, nothing is known about the possible elements that modulate its expression in fish. Our aim was to undertake the study of the possible endocrine regulation and the intracellular pathways involved in the gene expression of three noc paralogs (noc-a1, noc-a2 and noc-b1) expressed in goldfish (Carassius auratus). For this purpose, we studied the effect of glucagon, adrenaline and different activators and inhibitors of protein kinase A (PKA) and protein kinase C (PKC) pathways on noc paralogs expression in hepatopancreas organotypic cultures. We found a concentration-dependent induction of noc-a1 expression by both glucagon and adrenaline at 2 and 6-h incubation time. Forskolin, an adenylyl cyclase activator, induced a 5-fold increase of the relative abundance of this paralog, meanwhile a 2-fold increase was obtained in the presence of phorbol 12-myristate 13-acetate, a PKC activator. The effects of glucagon and forskolin were fully blocked by a PKA inhibitor (H89), and partially by a PKC inhibitor (chelerythrine) and by a PLC inhibitor (U73122), suggesting a possible interaction between PKA and PKC pathways in the intracellular signaling of glucagon on noc-a1 expression. Regarding noc-a2 mRNA expression, our results show an induction by adrenaline at 2 and 6-h incubation time but not by glucagon, forskolin or phorbol 12-myristate 13-acetate. Then, noc-b1 seems to be insensitive to both hormones, and to PKA and PKC activators. Altogether, these results suggest a possible functional specialization of noc paralogs in hepatopancreas and aim to investigate the possible role of other metabolic regulators in the control of noc expression in goldfish.
Supported by the Spanish MICINN (PID2019-103969RB-C32). D.M and A.A-G are predoctoral fellows, UCM (CT42/18-CT43/18) and FPI (BES-2017-081398), respectively.

The effects of simulated ocean acidification on the behaviour and physiology of the economically important Dungeness crab, Metacarcinus magister

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