Czechia

Balancing the control that is possible with lab studies with the real-world applicability of field work is a challenge in all experimental work. Using the example of the relationship between temperature, metabolic potential and social behaviour in fish, I will explore ways to incorporate more field relevance into lab studies and take lab-based theory to the field. As ectotherms, temperature has been called the “master factor” for fish; playing a large role in setting metabolic potential which in turn influences many aspects of behaviour and physiology. For example, changes in temperature have the potential to shift the cost-benefit analysis fish must conduct to balance the benefits of remaining in a group with the costs of increased competition. However, when considering the complexities of the natural world this relationship becomes less straight forward.

SEB Conference Prague 2024

Taking our understanding of thermal influences on social behaviour from the lab to the field

behaviour; thermal physiology; metabolism

technical paper

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Plasticity enables organisms to extend their tolerance limits, but the physiological basis of plastic responses is not well characterized. Here, we demonstrate that Drosophila melanogaster embryos undergo rapid thermal acclimation, on the timescale of hours, that extends their upper thermal limit. Because early development is coordinated by changes in chromatin state and gene activation, we set out to characterize the molecular basis of embryonic thermal plasticity by performing single nuclei multiome ATAC and RNA sequencing. We found coordinated changes in chromatin state and the transcriptome in response to thermal acclimation. Cool-acclimated embryos exhibited thermal compensation through increased chromatin accessibility of transcription factor binding motifs of global transcriptional activators and higher expression of genes encoding ribosomal proteins and enzymes involved in oxidative phosphorylation. Although most of these shifts were common to all cell types, many changes were cell type specific. For example, cold-acclimated embryos had higher overall gene expression in tracheal primordial cells, presumably to prepare for increased gas exchange to support a higher metabolic rate. On the other hand, we observed modestly higher expression of genes involved in cell migration and cell adhesion in warm-acclimated embryos, particularly in ventral nerve cord primordia, suggesting a potential role for locomotor and sensory development in embryonic heat tolerance. Our results indicate that thermal plasticity is largely mediated by shifts in the epigenome and transcriptome that regulate the speed of development and that may impose metabolic costs that constrain upper thermal limits.

Single nuclei multiome ATAC and RNA sequencing reveals the molecular basis of thermal acclimation in Drosophila melanogaster embryos

The Atlantic king scallop, Pecten maximus, as a subtidal species is more and more exposed to fluctuations in water temperature and pH variations due to the ongoing climate change. Its ability to swim makes scallops exceptional between bivalves and previous studies have shown that ocean warming (OW) and acidification (OA) can impact swimming performance of P. maximus. In particular, warming induced a decrease in the muscular index as well as a reduction in force and strength of the adductor muscle of P. maximus. However, the metabolic mechanisms behind these observed limitations in muscle performance are largely unexplored.
In this paper we present a semi-targeted metabolomic approach based on NMR spectroscopy for a retrospective study on frozen tissues of gill, mantle and adductor muscle of P. maximus, that were long-term acclimated under Ocean acidification, Ocean warming and the combination of both (OWA). 33 metabolites could be identified in all tissues, with organ depend differences in metabolite concentrations attributed to the specific functions of the individual organs. Metabolite changes to the different treatments showed that warming is the main driver for metabolic changes in gill and adductor muscle. 
In particular, energy-related metabolites, such as ATP and L-arginine, play an important role in the physiological response of scallops to OW and OWA.
With the help of a combined approach of pathway analysis and network exploration the warming induced changes in energy-related metabolites could be attributed to the observed limitations in swimming performance of P. maximus.

Metabolic profiling of muscle performance changes in the Atlantic king scallop, Pecten maximus, to ocean warming and acidification

The Northern shrimp (Pandalus borealis) is an ecologically and socio-economically important species in the North Atlantic, yet several of its different populations are collapsing due in part to global change drivers. Here, we aimed to elucidate the molecular mechanisms underlying shrimp responses to combined ocean warming (OW) and acidification (OA). Specifically, we profiled the phospholipidome of the abdominal muscle of female shrimp from four origins in the Northwest Atlantic, subjected to an orthogonal design of temperature (2, 6, or 10 °C) and pH (pH 7.75 or 7.40) scenarios. A suite of multivariate analyses revealed a complex interplay between shrimp origin and exposure to combined OW and OA, with OW primarily driving the phospholipidomic response, particularly at 10 °C. Warming to 10 °C altered phospholipids related to membrane thickness and lateral diffusion, and upregulated sphingomyelins. Notably, OA caused subtler effects, dependent on shrimp origin. Specimens from the Gulf of St. Lawrence showed an interactive effect of OW and OA, whilst those from the coasts of Newfoundland and Nova Scotia were not impacted by OA. Our finding has direct implications for prioritising conservation efforts on shrimp populations featuring a higher sensitivity to multiple stressors. Overall, our findings highlight the power of comparative omics to reveal different levels of intraspecific plasticity in the molecular response of marine organisms to future oceanic conditions. Our results will be refined by integrating multiomics and physiological traits within a multilayer framework to gain a comprehensive understanding of the response to global change drivers.

Molecular Responses of the Northern shrimp facing combined Ocean Warming and Acidification across the Northwest Atlantic Reveal Intraspecific Plasticity

In ectothermic species, changes in environmental conditions can be reflected in mitochondria, which are known for their high level of sensitivity and plasticity. These can result in either a functional change within the mitochondrion itself, a variation in total density within a tissue, or even a combination of the two. Therefore, experimental data must be presented with an adequate measure of mitochondrial density to be interpretable and scientifically backed. One of the most effective ways to quantify mitochondrial density (MD) is through transmission electron microscopy (TEM). However, proxies of mitochondrial density are often used in lieu. Commonly, quantifications of mitochondrial protein content, enzyme activity, and mitochondrial DNA copy numbers are employed as estimates of MD. The most frequently used estimates include measurements of citrate synthase (CS), complex I, and complex IV activity, but results vary greatly between studies. Indeed, mitochondrial plasticity, especially in ectotherms, may partly explain these discrepancies. This ongoing project therefore embodies a comprehensive approach evaluating if the use of mitochondrial density markers can be generalized in ectothermic animals. We show that despite cold-acclimated Drosophila (D. melanogaster) having higher complexe IV activity, and a tendency for increased CS activity, these commonly employed mitochondrial density markers were poor predictors of differences in various measurements of MD (i.e. number of mitochondria, fractional area, and surface density) evaluated by TEM. Conversely, neither differences were observed between enzymatic activities nor MD measurements between differentially acclimated beetles (Leptinotarsa decemlineata). The analysis of further density markers is ongoing.

Testing traditions: Validating mitochondrial density markers in ectothermic species

Ocean warming is one of the most pressing issues for marine species worldwide. Most marine species are ectothermic, and they rely on the conditions of the surrounding environment for appropriate development and reproduction. Thus, increasing temperatures usually lead to higher energetic requirements, and if these demands are not met, there can be cascading negative effects on key physiological processes. Further, ocean warming is also leading to the degradation of coral reef ecosystems, which is leading to a progressive simplification of tropical habitats. Despite recent advances in understanding how adaptation and plasticity (both developmental and transgenerational) can lead to compensation to environmental stressors, questions remain on the synergistic effect temperature and habitat simplification can have on marine species from both a molecular and physiological perspective. With this in mind, this seminar will focus on the molecular, behavioral and physiological responses to temperature fluctuations and habitat simplification in tropical fishes.

Transgenerational responses of marine fishes to ocean warming and habitat degradation 

Most studies exploring ectotherms metabolic response to warming focus on respirometric approaches, thus only reflecting individual aerobic metabolism. Here, we measured the metabolic response and lactate body content of Gammarus pulex after a shift from low (12°C) to high (22°C) temperature by coupled oxygen consumption measurements (microrespirometry) and heat production (direct microcalorimetry). Following the shift to high temperature and after an initial increase, oxygen consumption gradually decreases, getting back to the value reported at low temperature. Such phenotypic plasticity may compensate for the temperature-induced increase of metabolic rate, commonly considered as an acclimation process in ectotherms. However, the simultaneous measurements of Gammarus pulex metabolic rate by direct microcalorimetry remained stable, highlighting an uncoupling between organism heat production, reflecting total metabolic rate, and oxygen consumption. This mismatch suggests an oxygen-disconnected metabolic process, concomitant with an increase in individual lactate body content at high temperature, suggesting a shift from aerobic to anaerobic metabolism. Seven days after the shift to high temperature, the estimated proportion of anaerobic metabolic rate represents up to 50% of total metabolism. Our results indicate that the decrease in aerobic metabolism does not reflect a decrease in total metabolic rate, assimilated to metabolic acclimation, but rather a shift to anaerobic processes to sustain the temperature-induced increased in organism energetic requirements.

Metabolic acclimation: compensation or thermal stress?

There is global scientific and commercial interest in understanding the impacts of climate warming and heatwaves on the resilience of fish populations. While heat is thought to act at a fundamental level on biological reaction rates, protein structure and cell membrane fluidity, much has been made of the role of oxygen in governing these processes. The “gill-oxygen limitation” (GOL) hypothesis proposes that the growth of fish gills does not keep pace with the increase in metabolic requirements of the body as fish grow, resulting in a progressive mismatch between oxygen supply and demand that is exacerbated at high temperatures. We have empirically tested this hypothesis in thermally acclimated fish in recent years and found no supportive evidence; gills have the plasticity to grow proportionally with metabolism to ensure that oxygen supply and demand are matched as fish grow, even at elevated temperatures. We have, however, found some evidence for oxygen supply limitations across the gills during acute thermal exposures, exacerbated as fish increase in size. This talk will discuss the current state of knowledge on these topics with an aim to shed light on the mechanisms compromising fish resilience to acute heatwaves and chronic warming.

Potential for gill-oxygen limitation in acute but not chronic thermal tolerance

Lab-reared organisms allow researchers to understand aspects of our natural environment under controlled conditions contributing knowledge to policy development, risk assessment, and environmental systems. However, it is important to consider whether lab-reared organisms are actually representative models for their wild counterparts. To address this, our study compared the toxicity of ultraviolet filters (UVFs) between lab-reared and wild-caught Daphnia pulex to understand the effect of life-history on environmental exposures. 48-hour exposures of daphnids to avobenzone, oxybenzone, or octocrylene found minimal differences in both lethal and sublethal acute toxicity; however, several large deviations in chronic toxicity were observed over 21 day exposures. Wild daphnids exposed to 100 μg/L of octocrylene exhibited a 53% reduction in total reproduction compared to lab daphnids, as well as a 16% smaller core body size. In addition, differences in mortality were apparent between each population, as lab Daphnia experienced 30% higher mortality to 100 µg/L oxybenzone, while wild Daphnia experienced 30% higher mortality to 100 µg/L octocrylene. Overall, these results highlight a clear difference between the tolerance of lab-reared and wild organisms to UVFs, and that these differences are chemical specific. These results highlight the need for more environmentally relevant test methods for the use of model organisms across a multitude of fields, including physiology and toxicology. While the practicality and accessibility of lab-reared model organisms is an important tool, it is worth considering the use of their wild counterparts to provide a more accurate picture of these organismal functions in nature.

They're Sisters Not Twins: Understanding the differences in lab-reared & wild caught Daphnia pulex responses to ultraviolet filter exposure.

Suction-feeding is a common method for capturing prey by aquatic organisms. This method allows for the adaption of predators by enabling them to consume a wide range of prey. Suction- feeding is a complex fish-fluid interaction governed by various hydrodynamic forces: inertia, unsteady, viscous and pressure gradients that are described by the coupling between the flow physics equations (Navier-Stokes) and the dynamic behavior of the fish (motion and forces). This study focuses on estimating the pressure within the flow field surrounding the mouth of a bluegill sunfish (Lepomis macrochirus) during suction-feeding utilizing Particle Image Velocimetry (PIV). We used high-speed imaging for measurements of the fish buccal kinematics (period and amplitude). The pressure field is estimated from the velocity fields measured by PIV via the Poisson equation. The boundary conditions for the pressure field are determined from the integral momentum equation, separately, for each phase of the suction-feeding cycle. The spatial pressure distribution varied significantly as a function of phase. We explore the suction-feeding process by quantifying the pressure field that drives the flow towards the buccal cavity, where the location of a low-pressure zone varies throughout the feeding cycle.

Pressure field surrounding bluegill sunfish (Lepomis macrochirus) during suction-feeding

Formins are an evolutionarily old family of cytoskeletal regulators whose roles include microfilament capping, actin nucleation, and modulation of microtubule dynamics. Plant class I formins typically exhibit a unique domain organization, with most of them being transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space. Although such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins, they could also, at least temporarily, reside in endomembranes. Some of them decorate the plasmodesmata. We are currently developing techniques to use a photoswitchable fluorochrome for examining the effects of formin mutations on intercellular transport. Moreover, the main housekeeping Arabisopsis thaliana class I formin, AtFH1, relocates between the plasmalemma and several endomembrane compartments during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zone border. However, it is unclear whether the tonoplast-localized AtFH1 performs any biologically relevant function or whether is merely briefly occurs at the tonoplast en route towards vacuolar degradation. We shall present observations suggesting that both possibilities are mutually compatible: while mutational loss of AtFH1 affects tonoplast organization, the formin also enters the vacuole lumen. Another Class I formin, AtFH5, also enters the vacuole via an autophagic pathway under certain conditions and contributes to specific stress resistance. We propose a model where class I formins may serve as “active cargoes” of membrane trafficking, i.e. membrane-embedded proteins that modulate the fate of membrane compartments they reside on.
This work has been supported by the Czech Science Foundation grant 22-33471S.


Taking our understanding of thermal influences on social behaviour from the lab to the field

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Single nuclei multiome ATAC and RNA sequencing reveals the molecular basis of thermal acclimation in Drosophila melanogaster embryos

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Taking our understanding of thermal influences on social behaviour from the lab to the field

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Next from SEB Conference Prague 2024

Single nuclei multiome ATAC and RNA sequencing reveals the molecular basis of thermal acclimation in Drosophila melanogaster embryos

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