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Climate change has emerged as a critical issue in recent years. Previous research has highlighted the increase in prevalence of Vibrio in marine environments due to rising sea surface temperatures. This rise in pathogenic Vibrio has led to increased infection rates among humans and aquatic organisms. In freshwater ecosystems, Aeromonas stands out as the predominant pathogen. Both Vibrio and Aeromonas are known for their ability to thrive in diverse aquatic environments, posing significant health risks to both aquatic life and human populations. Therefore, understanding the dynamics of these pathogens in response to environmental changes is crucial for developing effective strategies to mitigate their impact on public health and ecosystem.
Our study primarily focuses on investigating Aeromonas, a facultative anaerobic Gram-negative bacillus commonly found in freshwater and wastewater environments. Aeromonas poses significant health risks to freshwater fish and can also lead to diseases in human, including diarrhea, wound infections, skin infections, pneumonia, and sepsis. Multiple Aeromonas isolates were gathered and subjected to strain typing via Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) techniques. Furthermore, we explore the antibiotic resistance profiles of Aeromonas isolates to understand the dynamics of resistance development. Bacteriophages were screened from various freshwater sources. Once corresponding phages were identified, they were purified for further investigations. 
These investigations offer potential applications in aquaculture to improve fish survival rates or in medical contexts to mitigate the pathogenic effects of Aeromonas on human disease.

SEB Conference Prague 2024

Phage-Based Eradication of Aeromonas: A Novel Approach against Aquatic and Clinical Pathogens

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The gills of farmed Atlantic salmon are susceptible to damage from various stressors, including toxins released by harmful algae (HA). Microcystin is a HA-toxin that affects fish and typically induces toxicity through oxidative stress with detoxification mainly occurring in the liver by glutathione (GSH). While the effect of microcystin on liver is well studied, its impact on gills, which are the first point of contact with microcystin has been overlooked. In this study, we explored the toxic effects of microcystin-LR (MC-LR) on the Atlantic salmon gill epithelial cell line (ASG-10) and whether N-acetyl-l-cysteine (NAC), a precursor of GSH, decreases these potential impacts.
The ASG-10 cells were exposed to different concentrations of MC-LR (0, 5, 10, 20, 40 µg/ml) in presence or absence of NAC (1 mM). Cell viability, reactive oxygen species (ROS) production, GSH level, and cell migratory behaviour/wound healing, were measured at different time points for up to 5 days post-exposure.
Cell death was only significantly increased after treatment with 40 µg/ml MC-LR after 5 days. There was a significant negative impact of MC-LR on proliferative and migratory ability of cells and the gap in the cell monolayer did not close when treated with 20 and 40 µg/ml of MC-LR. Interestingly, MC-LR had no impact on ROS production and GSH level in ASG-10 cells. However, NAC treatment was associated with reduced ROS and increased intracellular GSH as expected. These data indicate that MC-LR has negative effects on cell proliferation and may impair gill tissue healing and regeneration following damage.


The effect of harmful algal toxin on the Atlantic salmon gill epithelial cell line (ASG-10)

Natural vibrations are used by animals to communicate, to sense predators, detect prey, forage and reproduce. Vibrational communication assists both invertebrates and vertebrates to retrieve information from their surrounding environment. However, anthropogenic activities that are in direct contact with the seabed (such as pile-driving and mining) produce both sound in the water column and vibrations in the seabed. Exposure to vibrational noise has the potential to harm bottom-dwelling species in a similar way to water borne noise, potentially eliciting behavioural or physiological changes, or even causing physical damage. Research regarding substrate vibrations and marine invertebrates is in its infancy, with the vibrational sensitivities of most marine species relatively unknown; there is therefore a pressing need to understand the use of vibrations, since their usage defines how animals interact with their environment, as well as how human activity might interfere with natural biological processes. Here, we outline our project plan which will utilise different experimental setups to investigate the sensitivities of marine invertebrates to substrate-borne vibration. Preliminary studies will be done under controlled laboratory conditions to compare vibrational sensitivities, between and within taxa, through repeatable and reproducible methodologies. Overall, we aim to understand the effects of vibrational noise on key marine invertebrates and highlight the need for a shift in underwater noise research, to recognise vibrational noise in addition to water-borne sound.

Establishing methodologies for testing the sensitivity of marine invertebrates to substrate-borne vibrations

Microplastics (MPs), defined as plastic particles smaller than 5 mm in size, are considered a global environmental and human health problem, due to their ubiquity and persistence in the aquatic ecosystems. Considering the problematic of MPs, the present study aimed to assess the toxicological effects of MPs and copper (Cu) in zebrafish brain. Polyethylene (PE) and polystyrene (PS) MPs were selected because of their extensive commercial use and high accumulation in aquatic environments.
For this purpose, zebrafish were exposed for 21 days to Cu (25μg/L), PE and PS (1mg/L) and their mixtures (PE-Cu and PS-Cu). After 21 days, fish behavior was assessed, followed by their euthanasia with an overdose of buffered tricaine methanesulfonate, and excision of the whole brain for the biomarker’s evaluation. The biochemical parameters related to oxidative stress [Reactive oxygen species, lipid peroxidation]; antioxidant activity [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized states (GSSG) of glutathione levels]; and detoxification (glutathione-S-transferase) were assessed.
Data showed that the exploratory/locomotor, anxiety-related, and social behaviors were not significantly affected by Cu, PE, PS, and the respective mixtures. Considering the biochemical parameters, the GPx activity was significantly decreased in PE-Cu, in relation to the control group.
Considering our findings, the long-term behavioral and biochemical alterations in response to heavy metals and MPs, and its implications in fish population fitness still need to be clarified.

This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020.

The effect of polyethylene and polystyrene microplastics combined with copper in behavior and biochemical biomarkers of zebrafish (Danio rerio) brain

Several snake species sport horn-like appendages over their snouts and/or eyes. Although these structures have recently attracted scientific attention, their functional significance remains unknown. One hypothesis holds that the cephalic appendages may work as heat radiators/dissipators, keeping temperature stable during sudden changes in heat load. Following this hypothesis, we expect that horns heat and cool at different rates than the rest of the head and have a stabilizing influence on brain temperature. To test this idea, we used water-filled 3D-printed head models of vipers, the snake family with the highest number and diversity in horned species. After validating our method, we compared the heating and cooling of an array of hornless and horned models in a temperature-controlled environment and monitored horn and head temperatures. Our preliminary results show that horns indeed heat and cool at different rates than the rest of the head. However, the brain region of models with and without horns heated and cooled at similar rates. Therefore, unless they are accompanied by a special blood vessel cooling and countercurrent exchange system, the horn-like structures of snakes probably cannot function to regulate head/brain temperature. We are currently investigating the possibility of such rete mirabile in snakes, using micro-CT scanning and by monitoring heating and cooling behaviour of snakes in-vivo.

Thermal properties of horns in vipers - A preliminary but innovative study using 3D-printed models

While microplastics (MPs) have become the focus of an increasing amount of research in the last decade, there is still limited knowledge on the effects these pollutants have on the physiology of aquatic organisms. The aim of this study was to determine how exposure to polyethylene (PE) microplastics (38-42 µm, 250µg/L) impacts the physiology of two representative fish species; zebrafish (Danio rerio) and killifish (Fundulus heteroclitus) under different conditions. Behavioral changes and ammonia excretion rates were compared between zebrafish exposed to MPs and controls. Killifish were examined during acclimation to changing salinity with and without the added stress of MPs exposure. Zebrafish exposed to MPs showed a significant increase in ammonia excretion rates (p=0.042) compared with controls. Behavioral changes due to MPs exposure varied, showing significant differences in surfacing (p=0.021) and hyperventilation (p=0.036) behaviors. During salinity acclimation of killifish, MPs exposure eliminated the normal increase in drinking rates commonly seen in euryhaline fishes moving into higher salinity. This type of effect would have a significant impact on the capacity of killifish to rapidly acclimate to changing salinity. 


Impact of microplastics exposure on the physiology of zebrafish and killifish

Hummingbirds have unique physiological and morphological traits to fly at high speeds and hover in place. These adaptations require a high rate of metabolism, which generates body heat as a byproduct that must be dissipated. Dissipating heat can become increasingly challenging when the thermal gradient between the bird and surrounding air becomes smaller. I aim to characterize body surface temperature patterns across the hummingbird under different temperatures and flight durations. Since the feet are relatively uninsulated, we hypothesize that the feet region is a site for efficient thermal regulation during high metabolic activity. Anna’s hummingbirds (Calypte anna) were placed under four temperatures (20℃, 25℃, 30℃, 35℃) and flew for a period of 30, 60, 90, and 120 seconds. Thermal imaging was used to record surface body temperatures prior to and immediately after each exercise bout. We find that the feet have the greatest change (10℃ rise) between the lowest and highest temperature treatments, compared to other heat-dissipating areas such as the eyes and axial region which rise by 3-5℃. Moreover, the feet showed a slight increase in surface temperature as the flight duration increased at 30℃ and 35℃, while other areas did not change. Our results demonstrate that the feet are highly flexible thermoregulatory areas during post-exercise heat dissipation. Our future work will examine the bill’s importance, another poorly insulated region of the body.


The impact of exercise and high ambient temperature on heat dissipating areas in Anna's hummingbirds (Calypte anna)

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.

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

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. 


Phage-Based Eradication of Aeromonas: A Novel Approach against Aquatic and Clinical Pathogens

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The effect of harmful algal toxin on the Atlantic salmon gill epithelial cell line (ASG-10)

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