Czechia

Mussels from the Mytilus edulis species complex experience a salinity gradient from the North Sea into the Baltic Proper ranging from 32 to 5 practical salinity units. As osmoconformers, mussels adjust their internal osmolarity to match that of their surroundings. This adaptive strategy poses a significant challenge to the metabolic machinery of the mussels, including their mitochondria. Thus, populations from different sites have adapted to reach their highest mitochondrial function at a salinity close to their respective habitat salinity. We hypothesised that this adaptation is accompanied by a change in accumulated metabolites. To test this hypothesis, mussels from three populations along the salinity gradient were assessed: north Baltic Sea (M. trossulus zone), south Baltic Sea (transition zone) and North Sea (M. edulis zone). Metabolites from gill tissue were measured via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and their abundances analysed using a principal component analysis (PCA) and pathway enrichment analysis (MetaboAnalyst 6.0). The measured metabolites separated the populations from one another either by osmoregulation or by energy metabolism. Additionally, the pathway enrichment analysis identified three pathways that differed between each population. γ-aminobutyric acid (GABA) was one of the main drivers of the population specific differences, along with amino acids that are directly or indirectly connected to the citrate cycle. The citrate cycle in turn feeds the mitochondrial electron transport system (ETS). Overall, our findings suggest population specific differences in drivers of the citrate cycle as well as differences in the utilisation of sources for the mitochondrial respiration.

SEB Conference 2024

Metabolite profiling reveals energetic and osmoregulatory differences in populations from three zones of the Mytilus edulis species complex distribution area

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poster

The architectural properties of skeletal muscle provide an anatomical estimate of their functional capabilities. Gross dissection has long been the gold standard for exploring these variables, yet this technique has several inescapable drawbacks: being both irreversibly destructive and requiring the removal of a muscle from its original context for analysis thereby losing valuable spatial data. To counter these hurdles, several novel digital methodologies have emerged. Among these, diffusible iodine-based contrast-enhanced computed tomography (DiceCT) has been widely adopted to allow the mapping of individual fascicles in situ - enabling analysis of both their orientation and compression (i.e., tortuosity). Here, we present a cross-methodological analysis of four independent techniques for the reconstruction of muscle architectural variables: traditional gross dissection, manual segmentation of fascicles from DiceCT datastacks, and two distinct automated workflows (i.e., using Amira’s XFiber and the R package GoodFibes) using these same digital datasets. We applied these methods to a sample of eight mammals, ranging in body size from 0.1 to 10kg. First, we ran sensitivity analyses on our automated techniques to produce a single replicable workflow that could be applied across all taxa. Using this, we then calculated generally strong convergence in fascicle lengths between gross dissection, manual segmentation, and XFiber, while GoodFibes yielded consistently longer fascicles than would be expected. Both automated techniques also capture less fascicular tortuosity than manual fascicle segmentation. We conclude that automated methods represent a promising workflow to both enhance the speed of fascicular analysis and reduce user subjectivity and potential bias.

Multi-method analysis of three-dimensional techniques for the reconstruction of muscle architecture

Understanding how tiny organisms store and release energy through deformations of geometrically complex structures, such as the elastic head capsule of trap-jaw ants, is largely limited by the optical technology available to visualize small deformations across large surfaces within the mesoscale of living organisms (i.e., mm – cm). In this study, we develop an ultra-high-speed fourier light-field microscope capable of viewing specimens at high-resolution and frame rates of up to 1MHz+ (Photron E980S). In addition to validating the new instrument, we test its capabilities using Odontomachus brunneus trap-jaw ants, which can snap their mandibles shut in 0.08 ms, roughly 1000 times faster than the blink of an eye. These incredibly fast mandible strikes are possible through the slow loading and rapid unloading of elastic potential energy of the ant’s head. The head capsule deforms to store elastic potential energy during loading. During a strike, the head capsule recoils, transforming elastic potential energy stored through three dimensional deformations of the head into the rotational kinetic energy of the mandibles which move in a single plane. Using the new instrument, we successfully reconstruct the three-dimensional architecture of a portion of the head capsule during spring-loading and provide first measurements of real-time deformations in this portion of the head during unloading. These direct measurements of tiny deformations over larger fields in a structurally complex organism open a new realm for understanding energy flow and control in spring-propelled systems.

Visualization of tiny, springy deformations of an ultrafast trap-jaw ant's propulsive head

– Aposematism is a defensive strategy that combines a deterrent with visual signals. High intra-species variation in learning time to avoid the signal is seen, and this variation in behaviour influences prey defense evolution. Here, we aim to investigate if the micro-gut biome is a driver in maintaining this heterogeneity. Research with mammals establishes a relationship between gut microbiome, diet and behavior. We ask if this relationship impacts ecosystems and test if there is a feedback loop between the predator gut microbiome, cognition, foraging decisions and concentration of prey toxins consumed, and how this influences the selection pressures on chemical defenses. This provides multiple objectives: First, testing the association between the predator gut microbiome and foraging behaviour, quantifying the effect of consuming aposematic prey on the predator gut microbiome, identifying the defensive toxins that impact the predator gut microbiome, measuring the causal effect of the gut microbiome on behavior and then testing the effect of the early-life gut microbiome on foraging behaviour in adulthood. The model predators employed will be wild-caught great tits (Parus major), that will undergo altered gut microbiomes, through prey toxins and faecal microbiota transplantations. The microbial gut composition will be measured, and behavioral experiments will assess avoidance learning and dietary awareness. This investigation will deepen our understanding of the role of the predator gut microbiome in aposematism and uncover insights into the variation in predator behavior. Additionally, this investigation can propose if the gut microbiome is a factor in shaping ecology and evolution. 


The gut microbiome as a driver of foraging behaviour in predator-prey interactions.

Understanding how osmotic stress affects the performance of marine organisms is essential to produce sound predictions about the functioning of estuarine and coastal ecosystems with floods being more frequent and intense due to global changes. The green sea urchin Strongylocentrotus droebachiensis, an important top-down controller for coastal macroalgae, prefers salinities between 25 and 30; > 7 days at salinity 15 being its documented tolerance limit. Consequently, we aimed to characterize the effects of hypoosmotic stress on the physiological and behavioural responses of sea urchins during periods of flooding. We conducted a laboratory experiment along a gradient of six salinities (10, 13, 16, 20, 24 and 28 ppt), for one and two weeks. We evaluated survival, coordination performance of podia, podia adhesion, feeding rate, excretion rate, and coelomic fluid acid-base and osmotic balance following these exposure phases and following three days of recovery under control salinity conditions (28 ppt), . Urchins exposed to salinity 10 and 13 ppt experienced mortality throughout exposure, whilst survival was not compromised at salinities 16 to 28 ppt. Our results confirm an alteration of the physiological status of urchins via acid-base and osmoionic alterations and a reduction in feeding and excretion rates indicating behaviour modification. These imbalances were accompanied by a reduction in podia coordination and adhesion strength, which can lead to easier dislodgment due to waves or predators. This could ultimately lead to compromised survival and ecosystem consequences.

Characterization of the effect of hypoosmotic stress caused by coastal floodings on the physiology and behaviour of the Green Sea Urchin

Debate surrounds the role of behaviour in animal physiology, divided between traditional views that separate behaviour and physiology and a progressive perspective that supports the notion of behaviour as an inherent physiological effector. We advocate for the latter, and in this poster we provide a mechanistic and motivational perspective on behaviour and draw parallels with recognised physiological processes. Indeed, behaviour is ultimately generated by spatial and temporal patterns of musculoskeletal activity and it is tightly regulated by neural pathways, like other physiological processes. For example, many bird species will employ vasoconstriction and shivering – two well-established physiological processes in thermoregulation – and social huddling behaviour to avoid hypothermia. Yet, analogous neuroendocrine patterns can be observed in all three of these physiological actions. Furthermore, these responses are elicited by an animal’s motivational state in response to challenges in its environment. This motivational state can trigger a variety of physiological responses – behavioural or otherwise – that, while operating concurrently, all aim to satisfy an underlying ‘need’. For example, when an animal’s water balance approaches critical levels, it may employ a suite of physiological actions/strategies to conserve (e.g., water reabsorption in the gut, behavioural thermoregulation) and acquire water (e.g., water vapour absorption, drinking/feeding), driven by a motivational state to avoid osmotic stress. Our goal with this poster is to set the stage for a debate and persuade physiologists of the merits of including behaviour within the domain of animal physiology. Recognising behaviour as a physiological process is crucial for advancing a unified understanding of physiology.

Scaling organismal physiology: a case for the integration of behaviour as a physiological process

An important trait of Pacific bluefin tuna (PBT) is its ability to maintain body temperature through high metabolic heat production and its retention ability. PBT rapidly develop the thermo-conservation ability between 20 and 40 cm fork length (FL), which has been attributed to improved insulation. Meanwhile, how heat production capacity develops during ontogeny is not well understood. The aim of the present study was to explore the ontogenetic change in metabolic heat and its contribution to endothermic ability through swim-tunnel respirometry and a heat-budget model. Swim-tunnel respirometry was conducted on PBT, ranging from 19.4 to 27.5 cm FL (94–330 g, n=16) in Kochi Prefecture, Japan, from August to September 2022 and 2023. The metabolic rate of each PBT was calculated as the standard metabolic rate (BSMR) at a swim speed of 0 cm/s and the minimum metabolic rate (BUmin) at a minimum swim speed of 44.3 cm/s. The scaling exponents of BSMR and BUmin were estimated at 1.03 and 0.93, respectively. Both scaling exponents of metabolic rates were higher than those estimated for the tribe Thunnini above 300 g. Furthermore, a scaling exponent close to 1 implies that heat production rate (Tm in °C/min) in heat-budget model does not decrease in this body size range. To explore the ontogenetic change of heat-production rate of PBT in nature, heat-budget model was applied to bio-logging data collected from nine juveniles (size of analysis: 23–60 cm FL; 200–5272 g) with electronic archival tags inserted into their body cavity. The heat-production rate of each PBT did not decrease until approximately 800 g and declined thereafter, supporting the results of swim-tunnel respirometry. Furthermore, we evaluated the effects of heat production rate on the thermo-conservation ability, the difference between body temperature and water temperature, and found that maintaining a high heat production rate mainly contributed to the initial stage of temperature difference generation. Our results provided new insights into the endothermal development of tuna.

Highly maintained heat production capacity contributes to the initial step of endothermal development in Pacific bluefin tuna

A great proportion of people with Rett syndrome (RTT) have aberrant pain responses. However, due to additional RTT-related symptoms such as mobility impairments and altered verbal skills, it is difficult to assess their noxious responses. Apart of being considered a pathological state per se, altered pain responses could result in undertreatment of additional RTT-associated conditions. Here, we used male mice null for Mecp2, the gene causing most cases of RTT, to evaluate their response to an inflammatory model. At symptomatic ages (8 weeks old), two cohorts of animals received an intraplantar injection of Complete’s Freud Adjuvant. We analysed either mechanical or thermal sensitivity (von Frey hairs or Hargreaves apparatus, respectively) during basal conditions and at 1- and 3-days post-injection. An hour after last testing, animals were sacrificed and nervous tissues collected for testing. Controls included wild-type (WT) siblings and saline-injected animals from both genotypes. During basal conditions, Mecp2-null mice showed a significantly higher threshold to withdraw their paws in response to a mechanical stimulation than WT and thus are described as mechanically hyposensitive. Interestingly, although hypersensitivity also developed in the mutants at 24 hours post-injection, it was completely resolved by the third day. Immunofluorescence in sectioned brains revealed alterations in the endocannabinoid system within the Mecp2-null mice when compared to WT. Taking all together, our data suggests that MeCP2 is key player in the maintenance, but not the development, of mechanical hypersensitivity following an injury. We also point at the endocannabinoid system as a potential therapeutic target for RTT

Mecp2-null male mice are mechanically hyposensitive before and after an inflammatory pain model

Extreme heat events are predicted to be more frequent and intense over the next few decades. Ectotherms are particularly sensitive to such events due to their reliance on the external environment to perform their daily activities and maintain body temperature. Exposure to higher temperatures during fish development can accelerate growth rates, which directly impacts growth and mortality rates. Yet very little is known about the impact of rearing density and social cues on the behavior of developing embryos exposed to thermal stress. In this study, we measured the startle response of 1 day post fertilization zebrafish embryos at different rearing densities (10, 20, and 40 eggs), and with and without regular social and heat-induced chemical cues. Our results show that activity increases with higher embryo densities, and heat-induced social cues modulate activity both with and without application of thermal stress. Since the startle response is a proxy for how well sensory information and motor actions are coordinated, rearing density and social cues emerge as important factors for understanding the response of developing fish to environmental stressors.

Exploring Zebrafish Embryo Behavioral stress Responses in social contexts

Rett syndrome (RTT) is a neurodevelopmental disorder characterised by, among others, motor impairments, neuroendocrine dysregulation, anxiety altered responses and aberrant pain perception. The primary cause of RTT is de novo loss-of-function mutation in the methyl-CpG-binding domain protein 2 (MECP2) gene, which is located within chromosome X. Although RTT affects mainly females (heterozygous), most rodent studies have used males (hemizygous) as they develop the RTT-associated symptomatology sooner than females (8 weeks vs. 6 months). Here, we analysed both mutant males and females from a CD1 background line at corresponding symptomatic ages, together with control wild-type (WT) siblings. After weighting the animals, they were subjected to an elevated plus maze (EPM) to assess their anxiety responsiveness. One week later, animals received an intraplantar injection of capsaicin to elicit a nociception-specific activation. One hour post-injection, subjects were sacrificed and nervous system collected to assess differential activation in the nociceptive pathway via cFOS immunohistochemistry. Our results show that, although Mecp2 mutant males moved less over the duration of the EPM, they demonstrate a more anxiolytic phenotype than their WT siblings. Mecp2-het females seem to describe a similar behavioural pattern. Nonetheless, mutant females were significantly heavier than their WT siblings while males showed no difference in weight. Following the capsaicin injection, similar changes were observed in both males and females. Together, our results validate this rodent model for the study of RTT-associated neurological manifestation and further demonstrate the need to include animals from both sexes to study the aetiology of RTT and associated disorders.

Mecp2-mutant CD1 mice recapitulate anxiety- and nociceptive-alterations associated with Rett Syndrome

Mice rely on olfactory and vomeronasal marks to recognise other individuals, which need to be integrated into the spatial map to encode the identity of other conspecifics in a context-dependent manner. Our objective is to explore the influence of vomeronasal information in the spatial map characterising the functional interconnection between the chemosensory amygdala and the hippocampus.
We assessed vomeronasal influence on spatial mapping by presenting male urine to female mice in two exploratory behaviours while recording single-unit activity in the hippocampus. In one experiment, mice explore a virtual labyrinth divided into sectors with specific visual or olfactory contingencies. In the other, we designed a test to study place preference and spatial maps simultaneously, the Enriched Open Field (EOF), where we placed modular pieces in an Open Field to enhance exploration and provide references, and including urine marks in a specific quadrant.
The neuronal activity associated with the exploration of the labyrinth showed specific responses to given sectors. These responses include both increased and decreased firing rate usually in the sector where male urine is delivered. In the EOF, in contrast to our expectations, female mice avoid the area where the male left marks. But regarding the neuronal activity, we could identify neurons whose response is associated with male-marked areas.
These experiments broaden our knowledge about how neuronal activity links both exploration patterns and specific locations in space in the context of the integration of social cues. 


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Multi-method analysis of three-dimensional techniques for the reconstruction of muscle architecture

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Salinity dependent mitochondrial respiration in mussels of the Mytilus edulis species complex